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Liu Y, Raymond O, Hodgkiss JM. Exploring Fluorescence Spectral Shifts in Aptamer-Intercalating Cyanine Dye Complexes upon Binding to Specific Small Molecules. ACS Sens 2025; 10:2266-2275. [PMID: 39999296 DOI: 10.1021/acssensors.4c03579] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/27/2025]
Abstract
DNA intercalating cyanine dyes, such as SYBR Green I (SG) and OliGreen (OG), are widely used in developing label-free, fluorescent aptamer-based biosensors. Despite their widespread use for direct analyte detection through changes in fluorescence intensity, the effects of dye concentrations and the specific nature of their interactions have been inadequately explored. Here, we reported how dye-to-base ratios (dbrs) influence the fluorescent response of DNA intercalating dyes in aptamer systems targeting adenosine triphosphate (ATP) and l-argininamide (LAA). We initially examined the fluorescence spectral shifts of an ATP aptamer (ABA) with SG across varying dbrs, observing an emission shift to longer wavelengths as the dbrs increased. Subsequently, systematic analysis of the ATP aptamer and SG complex (ABA/SG) at different target concentrations revealed a "signal-off" phenomenon at a very low dbr of 0.1, which transitioned to a blue shift in the fluorescence spectra at higher dbr values of 0.7 and 2.0. Further extending our research, we explored the use of OG as a ratiometric probe for detecting l-argininamide, noting similar spectral shifts to shorter wavelengths upon target binding. Absorption spectroscopy, circular dichroism (CD), and meticulously designed control studies were employed to elucidate the spectral shift phenomenon comprehensively. Our findings underscore the significant impact of dye selection and concentration on the performance of fluorescence aptasensors and demonstrate that clear spectral shifts, indicative of target binding, occur upon binding to targets, particularly at higher dye loading; however, excessive dye concentrations can perturb the aptamer structure, reducing its binding affinity. We believe that our findings will provide new insights into designing aptamer-based fluorescence assays for the sensitive and specific detection of small molecules.
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Affiliation(s)
- Yasmin Liu
- MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington 6140, New Zealand
- School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington 6140, New Zealand
- Forensic Research & Development Department, Institute of Environmental Science and Research, PO Box 50348, Porirua 5240, New Zealand
| | - Onyekachi Raymond
- Forensic Research & Development Department, Institute of Environmental Science and Research, PO Box 50348, Porirua 5240, New Zealand
| | - Justin M Hodgkiss
- MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington 6140, New Zealand
- School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington 6140, New Zealand
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2
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Tong W, Han Y, Wang T, Wan J, Ma F, Zhang CY. Bidirectional Polymerization-Transcription Amplification-Encoded Dual-Color Fluorescent Biosensor for Label-Free and Primer-Free Detection of Multiple piRNAs. Anal Chem 2024. [PMID: 39250656 DOI: 10.1021/acs.analchem.4c03773] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/11/2024]
Abstract
PIWI-interacting RNAs (piRNAs) are a type of endogenous noncoding RNAs with a length of 24-31 nucleotides, and they can specifically bind with PIWI proteins to form the piRNA/PIWI complexes for regulating multiple physiological and pathological processes. Herein, we develop a bidirectional polymerization-transcription amplification-encoded dual-color fluorescent biosensor for label-free and primer-free measurements of multiple piRNAs. The designed hairpin probe contains a palindromic tail, and it can serve as the target recognition unit, polymerization primer, and transcription template. In the presence of target piRNAs, the hairpin probes are opened to expose a palindromic sequence that can trigger bidirectional polymerization and transcription reaction with the assistance of KF polymerase and T7 RNA polymerase for the production of numerous RNA aptamers. The aptamers subsequently bind with the corresponding fluorophores (DFHBI-1T/MG) to form the RNA aptamer-fluorophore complexes for the generation of enhanced fluorescence signals. This biosensor can sensitively detect piR-36026 with a limit of detection (LOD) of 82.08 aM and piR-36743 with a LOD of 44.44 aM. Moreover, it can quantify cellular piRNAs with single-cell sensitivity and distinguish cancer cells from normal cells. Furthermore, it has the capability of distinguishing the expression of piRNAs in the tissues of breast cancer patients and healthy individuals. By simply altering the target recognition site of the hairpin probe, this biosensor can be extended to detect various piRNAs, offering a powerful platform for piRNA-related clinical diagnostics and therapeutics.
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Affiliation(s)
- Weijie Tong
- School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing 211189, China
| | - Yun Han
- School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing 211189, China
| | - Tao Wang
- Department of Thoracic Surgery, Nanjing Drum Tower Hospital, Medical School, Nanjing University, Nanjing 210000, China
| | - Jiayi Wan
- School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing 211189, China
| | - Fei Ma
- School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing 211189, China
| | - Chun-Yang Zhang
- School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing 211189, China
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3
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Jeung JH, Han H, Jang SH, Lee CY, Ahn JK. One-pot, one-step, label-free miRNA detection method based on the structural transition of dumbbell probe. Talanta 2024; 274:125944. [PMID: 38537347 DOI: 10.1016/j.talanta.2024.125944] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Revised: 02/15/2024] [Accepted: 03/16/2024] [Indexed: 05/04/2024]
Abstract
In this study, we present a one-pot, one-step, label-free miRNA detection method through a structural transition of a specially designed dumbbell-shape probe, initiating a rolling circle transition (RCT). In principle, target miRNA binds to right loop of the dumbbell probe (DP), which allows structural change of the DP to circular form, exposing a sequence complementary to the T7 promoter (T7p) previously hidden within the stem. This exposure allows T7 RNA polymerase to initiate RCT, producing a repetitive Mango aptamer sequence. TO1-biotin, fluorescent dye, binds to the aptamer, inducing a detectable enhancement of fluorescence intensity. Without miR-141, the DP stays closed, RCT is prevented, and the fluorescence intensity remains low. By employing this novel strategy, target miRNA was successfully identified with a detection of 73 pM and a dynamic linear range of 0-10 nM. Additionally, the method developed enables one-pot, one-step, and label-free detection of miRNA, demonstrating potential for point-of-care testing (POCT) applications. Furthermore, the practical application of the designed technique was demonstrated by reliably detecting the target miRNA in the human serum sample. We also believe that the conceived approach could be widely used to detect not only miRNAs but also diverse biomolecules by simply replacing the detection probe.
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Affiliation(s)
- Jae Hoon Jeung
- Material & Component Convergence R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, Republic of Korea; Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, 05029, Republic of Korea
| | - Hyogu Han
- Material & Component Convergence R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, Republic of Korea; Department of Chemistry, Gangneung-Wonju National University, Gangneung, 25457, Republic of Korea
| | - Se Hee Jang
- Material & Component Convergence R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, Republic of Korea; Department of Medical Device Engineering and Management, College of Medicine, Yonsei University, Seoul, 03722, Republic of Korea
| | - Chang Yeol Lee
- Bionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
| | - Jun Ki Ahn
- Material & Component Convergence R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, Republic of Korea.
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Ruengdit C, Punyamung M, Intasai N, Pornprasert S. Single-tube multiplex real-time PCR with EvaGreen and high-resolution melting analysis for diagnosis of α0-thalassemia--SEA,--THAI, and--CR type deletions. PLoS One 2023; 18:e0293838. [PMID: 37930985 PMCID: PMC10627449 DOI: 10.1371/journal.pone.0293838] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2023] [Accepted: 10/20/2023] [Indexed: 11/08/2023] Open
Abstract
Regions with a high prevalence of α-thalassemia (α-thal) require simple, rapid, and accurate tests for carrier screening and prenatal diagnosis. Diagnosis of multiple deletions in a single tube is necessary to clearly identify individuals with α0-thalassemia in the routine setting, especially in at-risk couples. Therefore, we aimed to develop a single-tube multiplex real-time PCR with EvaGreen and high-resolution melting (HRM) analysis for the identification of α0-thalassemia Southeast Asian (SEA), Thai and Chiang Rai (CR) type deletions. The results of the HRM analysis indicated that the amplified fragments from α0-thal--CR,--THAI,--SEA, and the wild-type α-globin gene had specific peak heights at mean melting temperature (Tm) values of 85.40°C, 86.50°C, 87.65°C, and 91.04°C, respectively. The frequencies of α0-thal--SEA,--THAI,--CR obtained from routine testing in 2,135 samples were 17.89%, 0.19% and 0.19%, respectively. This method would be useful for preventing Hb Bart's hydrops fetalis. Detection of multiple deletions in a single run is cost-effective, highly accurate and timesaving. This technique could enable wider α-thalassemia diagnosis in high prevalence areas and served as an example for thalassemia routine setting.
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Affiliation(s)
- Chedtapak Ruengdit
- Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
| | - Manoo Punyamung
- Associated Medical Sciences Clinical Service Center, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
| | - Nutjeera Intasai
- Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
| | - Sakorn Pornprasert
- Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
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Jiang S, Shi H, Zhang Q, Wang ZY, Zhang Y, Zhang CY. Rolling circle transcription amplification-directed construction of tandem spinach-based fluorescent light-up biosensor for label-free sensing of β-glucosyltransferase activity. Biosens Bioelectron 2023; 237:115513. [PMID: 37419074 DOI: 10.1016/j.bios.2023.115513] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Revised: 06/17/2023] [Accepted: 07/03/2023] [Indexed: 07/09/2023]
Abstract
β-glucosyltransferase (β-GT) can specifically catalyze the conversion of 5-hydroxymethylcytosine (5-hmC) to 5-glucosylhydroxy methylcytosine (5-ghmC), and it is associated with the control of phage-specific gene expression by affecting transcription process in vivo and in vitro. The current strategies for β-GT assay usually involve expensive equipment, laborious treatment, radioactive hazard, and poor sensitivity. Here, we report a Spinach-based fluorescent light-up biosensor for label-free measurement of β-GT activity by utilizing 5-hmC glucosylation-initiated rolling circle transcription amplification (RCTA). We design a 5-hmC-modified multifunctional circular detection probe (5-hmC-MCDP) that integrates the functions of target-recognition, signal transduction, and transcription amplification in one probe. The introduction of β-GT catalyzes 5-hmC glucosylation of 5-hmC-MCDP probe, protecting the glucosylated 5-mC-MCDP probe from the cleavage by MspI. The remaining 5-hmC-MCDP probe can initiate RCTA reaction with the aid of T7 RNA polymerase, generating tandem Spinach RNA aptamers. The tandem Spinach RNA aptamers can be lightened up by fluorophore 3,5-difluoro-4-hydroxybenzylidene imidazolinone, facilitating label-free measurement of β-GT activity. Notably, the high specificity of MspI-catalyzed cleavage of nonglucosylated probe can efficiently inhibit nonspecific amplification, endowing this assay with a low background. Due to the higher efficiency of RCTA than the canonical promoter-initiated RNA synthesis, the signal-to-noise ratio of RCTA is 4.6-fold higher than that of linear template-based transcription amplification. This method is capable of sensitively detecting β-GT activity with a limit of detection of 2.03 × 10-5 U/mL, and it can be used for the screening of inhibitors and determination of kinetic parameters, with great potential in epigenetic research and drug discovery.
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Affiliation(s)
- Su Jiang
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China
| | - Huanhuan Shi
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China
| | - Qian Zhang
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China
| | - Zi-Yue Wang
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China.
| | - Yan Zhang
- College of Chemistry and Chemical Engineering, Qilu Normal University, Jinan, 250200, China.
| | - Chun-Yang Zhang
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China; School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China.
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6
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Baral B, Nial PS, Subudhi U. Enhanced enzymatic activity and conformational stability of catalase in presence of tetrahedral DNA nanostructures: A biophysical and kinetic study. Int J Biol Macromol 2023; 242:124677. [PMID: 37141969 DOI: 10.1016/j.ijbiomac.2023.124677] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2023] [Revised: 04/07/2023] [Accepted: 04/27/2023] [Indexed: 05/06/2023]
Abstract
The emergence of DNA nanotechnology has shown enormous potential in a vast array of applications, particularly in the medicinal and theranostics fields. Nevertheless, the knowledge of the compatibility between DNA nanostructures and cellular proteins is largely unknown. Herein, we report the biophysical interaction between proteins (circulatory protein bovine serum albumin, BSA, and the cellular enzyme bovine liver catalase, BLC) and tetrahedral DNA (tDNAs), which are well-known nanocarriers for therapeutics. Interestingly, the secondary conformation of BSA or BLC was unaltered in the presence of tDNAs which supports the biocompatible property of tDNA. In addition, thermodynamic studies showed that the binding of tDNAs with BLC has a stable non-covalent interaction via hydrogen bond and van der Waals contact, which is indicative of a spontaneous reaction. Furthermore, the catalytic activity of BLC was increased in the presence of tDNAs during 24 h of incubation. These findings indicate that the presence of tDNA nanostructures not only ensures a steady secondary conformation of proteins, but also stabilize the intracellular proteins like BLC. Surprisingly, our investigation discovered that tDNAs have no effect on albumin proteins, either by interfering or by adhering to the extracellular proteins. These findings will aid in the design of future DNA nanostructures for biomedical applications by increasing the knowledge on the biocompatible interaction of tDNAs with biomacromolecules.
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Affiliation(s)
- Bineeth Baral
- DNA Nanotechnology & Application Laboratory, CSIR-Institute of Minerals & Materials Technology, Bhubaneswar 751013, Odisha, India; School of Biological Sciences, Academy of Scientific & Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Partha S Nial
- DNA Nanotechnology & Application Laboratory, CSIR-Institute of Minerals & Materials Technology, Bhubaneswar 751013, Odisha, India; School of Biological Sciences, Academy of Scientific & Innovative Research (AcSIR), Ghaziabad 201002, India
| | - Umakanta Subudhi
- DNA Nanotechnology & Application Laboratory, CSIR-Institute of Minerals & Materials Technology, Bhubaneswar 751013, Odisha, India; School of Biological Sciences, Academy of Scientific & Innovative Research (AcSIR), Ghaziabad 201002, India.
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7
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Zhao NN, Liu WJ, Tian X, Zhang B, Zhang CY. Target-activated cascade transcription amplification lights up RNA aptamers for label-free detection of metalloproteinase-2 activity. Chem Commun (Camb) 2023; 59:1058-1061. [PMID: 36606583 DOI: 10.1039/d2cc06784f] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
We demonstrate that target-activated cascade transcription amplification lights up RNA aptamers for label-free detection of metalloproteinase-2 (MMP-2) activity with zero background. This assay exhibits good specificity and high sensitivity with a limit of detection (LOD) of 0.6 fM. Moreover, it can analyze enzyme kinetic parameters, screen inhibitors, and accurately quantify MMP-2 in cancer cells and clinical serums.
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Affiliation(s)
- Ning-Ning Zhao
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China.
| | - Wen-Jing Liu
- School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
| | - Xiaorui Tian
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China.
| | - Baogang Zhang
- Department of Clinical Pathology, Affiliated Hospital of Weifang Medical University, Weifang Medical University, Weifang 261053, China.
| | - Chun-Yang Zhang
- College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China.
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8
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Baumann V, Athanasiou AT, Faridani OR, Schwerdtfeger AR, Wallner B, Steinborn R. Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma. Front Genet 2023; 13:1058668. [PMID: 36685854 PMCID: PMC9846067 DOI: 10.3389/fgene.2022.1058668] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2022] [Accepted: 12/02/2022] [Indexed: 01/05/2023] Open
Abstract
We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% (p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%-65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%-95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18-55 years). In general, inter-individual variance of miRNA abundance was low or very low as indicated by coefficient of variation (CV) values of 0.6%-8.2%. miR-3665 and miR-1915-3p outperformed in this analysis (CVs: 0.6 and 2.4%, respectively). The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor (NF) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes.
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Affiliation(s)
- Volker Baumann
- Genomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, Austria
| | | | - Omid R. Faridani
- Garvan Institute of Medical Research, Sydney, NSW, Australia,Lowy Cancer Research Centre, School of Biomedical Sciences, University of New South Wales, Sydney, NSW, Australia
| | | | - Bernard Wallner
- Department of Behavioral and Cognitive Biology, University of Vienna, Vienna, Austria
| | - Ralf Steinborn
- Genomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, Austria,Department of Microbiology, Immunobiology and Genetics, University of Vienna, Vienna, Austria,*Correspondence: Ralf Steinborn,
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9
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Enespa, Chandra P. Tool and techniques study to plant microbiome current understanding and future needs: an overview. Commun Integr Biol 2022; 15:209-225. [PMID: 35967908 PMCID: PMC9367660 DOI: 10.1080/19420889.2022.2082736] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/02/2022] Open
Abstract
Microorganisms are present in the universe and they play role in beneficial and harmful to human life, society, and environments. Plant microbiome is a broad term in which microbes are present in the rhizo, phyllo, or endophytic region and play several beneficial and harmful roles with the plant. To know of these microorganisms, it is essential to be able to isolate purification and identify them quickly under laboratory conditions. So, to improve the microbial study, several tools and techniques such as microscopy, rRNA, or rDNA sequencing, fingerprinting, probing, clone libraries, chips, and metagenomics have been developed. The major benefits of these techniques are the identification of microbial community through direct analysis as well as it can apply in situ. Without tools and techniques, we cannot understand the roles of microbiomes. This review explains the tools and their roles in the understanding of microbiomes and their ecological diversity in environments.
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Affiliation(s)
- Enespa
- Department of Plant Pathology, School of Agriculture, SMPDC, University of Lucknow, Lucknow, India
| | - Prem Chandra
- Department of Environmental Microbiology, Babasaheb Bhimrao Ambedkar (A Central) University, Lucknow, India
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Selection and identification of a DNA aptamer for fluorescent detection of netilmicin. Talanta 2022; 250:123708. [PMID: 35752088 DOI: 10.1016/j.talanta.2022.123708] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2022] [Revised: 05/26/2022] [Accepted: 06/16/2022] [Indexed: 11/22/2022]
Abstract
Netilmicin (NET) is an antibiotic widely used in healthcare and agriculture, but it can accumulate in the environment to threat human health. Netilmicin (NET) is an antibiotic used for veterinary purposes, for human therapy and for agricultural purposes. Therefore, there is a need to develop high-sensitive measuring methods to detect NET. Aptamer-based detecting methods are highly sensitive, inexpensive, and portable. In this study, we developed an aptamer-based fluorescence method to detect and quantify NET. NET was first conjugated to magnetic beads by amidation reaction and then NET-coated beads were used as the stationary phase to isolate aptamers by systematic evolution of ligands by exponential enrichment (SELEX) screening method. After ten rounds of SELEX screening, 32 aptamers with NET-binding affinity were obtained and the candidate aptamer APT-21 was finally chosen by comprehensively comparing their secondary structure characters and NET-binding affinity. APT-21 bound to NET with high affinity (Kd = 194.1 nmol/L) and high specificity that it displayed low cross-binding activities on 7 different structural analogs. We also developed a fluorometric assay using SYBR Green I (SG-I) and the APT-21. Key experimental parameters were optimized, including buffer system, SG-I and APT-21 reaction time, SG-I concentration, and aptamer concentration, to improve the detecting sensitivity. Our results suggest that the low limit of detection (LOD) of this method reached a low level of 1.95 nM and it also exhibited a good linear range up to 200 nM. Moreover, we successfully applied our method to detect the NET spiked in tap water and river water with good recoveries in the range from 97% to 111%. In conclusion, our current study isolated a NET-specific aptamer and developed an aptamer-based quantification method, which is promising to apply to detect NET in environmental samples.
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11
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Fu H, Zhang C, Wang Y, Chen G. Advances in multiplex molecular detection technologies for harmful algae. ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH INTERNATIONAL 2022; 29:43745-43757. [PMID: 35449333 DOI: 10.1007/s11356-022-20269-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/02/2022] [Accepted: 04/11/2022] [Indexed: 06/14/2023]
Abstract
As the eutrophication of natural water bodies becomes more and more serious, the frequency of outbreaks of harmful algal blooms (HABs) mainly formed by harmful algae also increases. HABs have become a global ecological problem that poses a serious threat to human health and food safety. Therefore, it is extremely important to establish methods that can rapidly detect harmful algal species for early warning of HABs. The traditional morphology-based identification method is inefficient and inaccurate. In recent years, the rapid development of molecular biology techniques has provided new ideas for the detection of harmful algae and has become a research hotspot. The current molecular detection methods for harmful algal species mainly include fluorescence in situ hybridization, sandwich hybridization, and quantitative PCR (qPCR), but all of these methods can only detect single harmful algal species at a time. The establishment of methods for the simultaneous detection of multiple harmful algal species has become a new trend in the development of molecular detection technology because various harmful algal species may coexist in the natural water environment. The established molecular techniques for multiple detections of harmful algae mainly include gene chip, multiplex PCR, multiplex qPCR, massively parallel sequencing, antibody chip, and multiple isothermal amplification. This review mainly focuses on the principles, advantages and disadvantages, application progress, and application prospects of these multiple detection technologies, aiming at providing effective references not only for the fisheries but also for economic activities, environment, and human health.
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Affiliation(s)
- Hanyu Fu
- College of Oceanology, Harbin Institute of Technology (Weihai), Weihai, 264209, People's Republic of China
| | - Chunyun Zhang
- College of Oceanology, Harbin Institute of Technology (Weihai), Weihai, 264209, People's Republic of China
| | - Yuanyuan Wang
- College of Oceanology, Harbin Institute of Technology (Weihai), Weihai, 264209, People's Republic of China
| | - Guofu Chen
- College of Oceanology, Harbin Institute of Technology (Weihai), Weihai, 264209, People's Republic of China.
- School of Environment, Harbin Institute of Technology, Harbin, 150009, People's Republic of China.
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12
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Pearson K, Doherty C, Zhang D, Becker NA, Maher LJ. Optimized quantitative PCR analysis of random DNA aptamer libraries. Anal Biochem 2022; 650:114712. [PMID: 35561815 PMCID: PMC9542921 DOI: 10.1016/j.ab.2022.114712] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2022] [Revised: 04/29/2022] [Accepted: 05/02/2022] [Indexed: 11/19/2022]
Abstract
The quantitative polymerase chain reaction (qPCR) with detection of duplex DNA yield by intercalator fluorescence is a common and essential technique in nucleic acid analysis. We encountered unexpected results when applying standard qPCR methods to the quantitation of random DNA libraries flanked by regions of fixed sequence, a configuration essential for in vitro selection experiments. Here we describe the results of experiments revealing why conventional qPCR methods can fail to allow automated analysis in such cases, and simple solutions to this problem. In particular we show that renaturation of PCR products containing random regions is incomplete in late PCR cycles when extension fails due to reagent depletion. Intercalator fluorescence can then be lost at standard interrogation temperatures. We show that qPCR analysis of random DNA libraries can be achieved simply by adjusting the step at which intercalator fluorescence is monitored so that the yield of annealed constant regions is detected rather than the yield of full duplex DNA products.
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Affiliation(s)
- Keenan Pearson
- Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA; Biochemistry and Molecular Biology Track, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA
| | - Caroline Doherty
- Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA; Medical Scientist Training Program, Mayo Clinic Graduate School of Biomedical Sciences and Mayo Clinic Alix School of Medicine, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA
| | - Drason Zhang
- Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA
| | - Nicole A Becker
- Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA
| | - L James Maher
- Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 200 First St. SW, Rochester, MN, 55905, USA.
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Ruengdit C, Punyamung M, Khamphikham P, Pongpunyayuen P, Intasai N, Pornprasert S. Multiplex Quantitative Real-Time Polymerase Chain Reaction and High-Resolution Melting Analysis for Identification of a Couple At-Risk of Having a Newborn with Severe Thalassemia. Hemoglobin 2022; 45:309-313. [PMID: 35139748 DOI: 10.1080/03630269.2022.2028634] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
Many polymerase chain reaction (PCR)-based techniques have been used for routine diagnosis of α- and β-thalassemias. However, most require a multi step of post-PCR processes that are time-consuming and labor-intensive procedures. This study reported the successful use of multiplex quantitative real-time PCR (qPCR), with high-resolution melting (HRM) analysis for diagnosis of two common deletional α0-thalassemia (α0-thal) and 15 common β-thalassemia (β-thal) mutations, in order to identify a couple at-risk of having a newborn with severe thalassemia in the northern region of Thailand. With this approach, 22 (7.2%) of 306 couples were diagnosed as being at-risk for having a child with severe thalassemia, including three homozygous α0-thal, five homozygous β-thal and 14 Hb E (HBB: c.79G>A)/β0-thal disease. Our findings indicated that multiplex qPCR with HRM is applicable for routine molecular diagnosis in order to identify a couple at-risk of having a newborn with severe thalassemia, especially in an endemic region.
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Affiliation(s)
- Chedtapak Ruengdit
- Division of Clinical Microscopy, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
| | - Manoo Punyamung
- Associated Medical Sciences Clinical Service Center, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
| | - Pinyaphat Khamphikham
- Division of Clinical Microscopy, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
| | - Panida Pongpunyayuen
- Associated Medical Sciences Clinical Service Center, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
| | - Nutjeera Intasai
- Division of Clinical Microscopy, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
| | - Sakorn Pornprasert
- Division of Clinical Microscopy, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
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14
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Morphologic and genetic analysis for geographic populations of greenbug Schizaphis graminum (Hemiptera: Aphididae) in Egypt. Biologia (Bratisl) 2021. [DOI: 10.2478/s11756-020-00501-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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15
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Kim W, Yoon Y, Seo Y, Lee Y, Lee H, Kim S, Ha J, Choi Y, Oh H, Kim Y, Kang J, Park E, Yoo Y, Sung M, Lee S. Development of Listeria monocytogenes detection technique in mushroom based on real-time quantitative PCR through improvement of enrichment medium. FOOD SCIENCE AND TECHNOLOGY RESEARCH 2021. [DOI: 10.3136/fstr.27.837] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Affiliation(s)
- Woori Kim
- Department of Food and Nutrition, Sookmyung Women's University
| | - Yohan Yoon
- Department of Food and Nutrition, Sookmyung Women's University
| | - Yeongeun Seo
- Department of Food and Nutrition, Sookmyung Women's University
| | - Yewon Lee
- Department of Food and Nutrition, Sookmyung Women's University
| | - Heeyoung Lee
- Food Standard Research Center, Korea Food Research Institute
| | | | | | | | - Hyemin Oh
- Department of Food and Nutrition, Sookmyung Women's University
| | - Yujin Kim
- Department of Food and Nutrition, Sookmyung Women's University
| | - Joohyun Kang
- Department of Food and Nutrition, Sookmyung Women's University
| | - Eunyoung Park
- Department of Food and Nutrition, Sookmyung Women's University
| | - Yoonjeong Yoo
- Department of Food and Nutrition, Sookmyung Women's University
| | - Miseon Sung
- Department of Food and Nutrition, Sookmyung Women's University
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16
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Caprara D, Ripanti F, Capocefalo A, Ceccarini M, Petrillo C, Postorino P. Exploiting SERS sensitivity to monitor DNA aggregation properties. Int J Biol Macromol 2020; 170:88-93. [PMID: 33358955 DOI: 10.1016/j.ijbiomac.2020.12.039] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Revised: 11/19/2020] [Accepted: 12/05/2020] [Indexed: 01/11/2023]
Abstract
In the last decades, DNA has been considered far more than the system carrying the essential genetic instructions. Indeed, because of the remarkable properties of the base-pairing specificity and thermoreversibility of the interactions, DNA plays a central role in the design of innovative architectures at the nanoscale. Here, combining complementary DNA strands with a custom-made solution of silver nanoparticles, we realize plasmonic aggregates to exploit the sensitivity of Surface Enhanced Raman Spectroscopy (SERS) for the identification/detection of the distinctive features of DNA hybridization, both in solution and on dried samples. Moreover, SERS allows monitoring the DNA aggregation process by following the temperature variation of a specific spectroscopic marker associated with the Watson-Crick hydrogen bond formation. This temperature-dependent behavior enables us to precisely reconstruct the melting profile of the selected DNA sequences by spectroscopic measurements only.
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Affiliation(s)
- Debora Caprara
- Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
| | | | - Angela Capocefalo
- Istituto dei Sistemi Complessi-CNR c/o Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
| | - Marina Ceccarini
- National Centre for Rare Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
| | - Caterina Petrillo
- Physics and Geology Department, University of Perugia, Via A. Pascoli, 06123 Perugia, Italy
| | - Paolo Postorino
- Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
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17
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Kim S, Kim S. Bacterial pathogen detection by conventional culture‐based and recent alternative (polymerase chain reaction, isothermal amplification, enzyme linked immunosorbent assay, bacteriophage amplification, and gold nanoparticle aggregation) methods in food samples: A review. J Food Saf 2020. [DOI: 10.1111/jfs.12870] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Affiliation(s)
- Sang‐Oh Kim
- Department of Plant and Food Sciences Sangmyung University Cheonan Republic of Korea
| | - Sang‐Soon Kim
- Department of Food Engineering Dankook University Cheonan Republic of Korea
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18
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Azinheiro S, Carvalho J, Prado M, Garrido-Maestu A. Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula. Microorganisms 2020; 8:E1359. [PMID: 32899815 PMCID: PMC7564587 DOI: 10.3390/microorganisms8091359] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2020] [Revised: 09/02/2020] [Accepted: 09/03/2020] [Indexed: 01/23/2023] Open
Abstract
Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.
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Affiliation(s)
- Sarah Azinheiro
- Food Quality and Safety Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, Portugal; (S.A.); (J.C.); (M.P.)
- College of Pharmacy/School of Veterinary Sciences, University of Santiago de Compostela, Campus Vida, E-15782 Santiago de Compostela, Spain
| | - Joana Carvalho
- Food Quality and Safety Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, Portugal; (S.A.); (J.C.); (M.P.)
- College of Pharmacy/School of Veterinary Sciences, University of Santiago de Compostela, Campus Vida, E-15782 Santiago de Compostela, Spain
| | - Marta Prado
- Food Quality and Safety Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, Portugal; (S.A.); (J.C.); (M.P.)
| | - Alejandro Garrido-Maestu
- Food Quality and Safety Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, Portugal; (S.A.); (J.C.); (M.P.)
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19
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Zheng M, Lin S, Zhang S, Chen X, Jiang D, Chen S, Wang S, Chen S. Rapid detection of H146-like goose calicivirus using real-time RT-PCR with a Taqman minor groove binder probe. J Virol Methods 2020; 285:113956. [PMID: 32814077 DOI: 10.1016/j.jviromet.2020.113956] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2020] [Revised: 08/10/2020] [Accepted: 08/11/2020] [Indexed: 11/28/2022]
Abstract
H146-like goose-origin calicivirus (H146-like GCV) is a novel Caliciviridae family member in the Sanovirus genus that was associated with gosling growth retardation syndrome growth retardation syndrome complicated by visceral urate deposition. However, there is no accurate and high throughput real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) available for the rapid and highly sensitive identification of H146-like GCV. In this study, a pair of specific primers and a TaqMan minor groove binder (MGB) probe were designed based on a conserved region in the nonstructural (NS) gene sequence. The TaqMan-MGB probe-based one-step qRT-PCR assay was capable of detecting quite low number of targeting nucleic acid as low as 5.07 copies/μL and had excellent intra-assay and inter-assay repeatability with the coefficient of variation (CV) value from 0.558% to 1.293%. The assay was highly specific for H146-like GCV, without cross-reactions with other non-targeted goose-origin viruses, and 62 suspicious tissue samples infected with H146-like GCV from different regions of Fujian Province were used in this study to verify the feasibility and effectiveness of this assay in clinical diagnosis. The results indicated that our assay for the diagnosis and quantification of H146-like GCV was highly sensitive and specific, and should provide a reliable real-time tool for epidemiological and pathogenetic study of H146-like GCV infection, enabling researchers to better understand the epidemiology and clinical presentation of this disease.
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Affiliation(s)
- Min Zheng
- Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou 350003, China; Fujian Animal Diseases Control Technology Development Center, Fuzhou 350013, China
| | - Su Lin
- Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou 350003, China; Fujian Animal Diseases Control Technology Development Center, Fuzhou 350013, China
| | - Shizhong Zhang
- Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou 350003, China; Fujian Animal Diseases Control Technology Development Center, Fuzhou 350013, China
| | - Xiuqin Chen
- Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou 350003, China; Fujian Animal Diseases Control Technology Development Center, Fuzhou 350013, China
| | - Dandan Jiang
- Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou 350003, China
| | - Shaoying Chen
- Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou 350003, China; Fujian Animal Diseases Control Technology Development Center, Fuzhou 350013, China
| | - Shao Wang
- Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou 350003, China; Fujian Animal Diseases Control Technology Development Center, Fuzhou 350013, China.
| | - Shilong Chen
- Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou 350003, China; Fujian Animal Diseases Control Technology Development Center, Fuzhou 350013, China.
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20
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Quantitative detection of economically important Fusarium oxysporum f. sp. cubense strains in Africa in plants, soil and water. PLoS One 2020; 15:e0236110. [PMID: 32687514 PMCID: PMC7371176 DOI: 10.1371/journal.pone.0236110] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2020] [Accepted: 06/28/2020] [Indexed: 01/12/2023] Open
Abstract
Banana is an important food crop and source of income in Africa. Sustainable production of banana, however, is at risk because of pests and diseases such as Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). Foc can be disseminated from infested to disease-free fields in plant material, water and soil. Early detection of Foc using DNA technologies is thus required to accurately identify the fungus and prevent its further dissemination with plants, soil and water. In this study, quantitative (q)PCR assays were developed for the detection of Foc Lineage VI strains found in central and eastern Africa (Foc races 1 and 2), Foc TR4 (vegetative compatibility groups (VCG) 01213/16) that is present in Mozambique, and Foc STR4 (VCG 0120/15) that occurs in South Africa. A collection of 127 fungal isolates were selected for specificity testing, including endophytic Fusarium isolates from banana pseudostems, non-pathogenic F. oxysporum strains and Foc isolates representing the 24 VCGs in Foc. Primer sets that proved to be specific to Foc Lineage VI, Foc TR4 and Foc STR4 were used to produce standard curves for absolute quantification, and the qPCR assays were evaluated based on the quality of standard curves, repeatability and reproducibility, and limits of quantification (LOQ) and detection (LOD). The qPCR assays for Foc Lineage VI, TR4 and STR4 were repeatable and reproducible, with LOQ values of 10−3–10−4 ng/μL and a LOD of 10−4–10−5 ng/μL. The quantitative detection of Foc strains in Africa could reduce the time and improve the accuracy for identifying the Fusarium wilt pathogen from plants, water and soil on the continent.
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21
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Shen R, Jia Y, Mak PI, Martins RP. Clip-to-release on amplification (CRoA): a novel DNA amplification enhancer on and off microfluidics. LAB ON A CHIP 2020; 20:1928-1938. [PMID: 32352133 DOI: 10.1039/d0lc00318b] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
Despite its high sensitivity, low cost, and high efficiency as a DNA amplification indicator with a yes/no answer, dsDNA-binding dye encounters incompatibility when used in microfluidic systems, resulting in problems such as false negative amplification results. Besides, its inhibition of amplification at high concentrations hinders its application both on-chip and off-chip. In this study, we propose a novel DNA amplification enhancer to counteract the drawbacks of dsDNA-binding dyes. It acts as a temporary reservoir for the free-floating dyes in solution and releases them on demand during the amplification process. Through this clip-to-release on amplification mechanism, the enhancer lowered the background fluorescence of sample droplets before amplification, enhanced the signal-to-background ratio of positive samples, and eliminated the false negative signal of on-chip PCR. Moreover, the enhancer increased the off-chip polymerase chain reaction (PCR) efficiency, boosted the fluorescence signal up to 10-fold, and made less nonspecific amplification product. All the factors affecting the enhancer's performance are investigated in detail, including its structure and concentration, and the types of dsDNA-binding dye used in the reaction. Finally, we demonstrated the broad application of the proposed amplification enhancer in various DNA amplification systems, for various genes, and on various amplification platforms. It would reignite the utilization of dsDNA dyes for wider applications in DNA analysis both on-chip and off-chip.
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Affiliation(s)
- Ren Shen
- State-Key Laboratory of Analog and Mixed-Signal VLSI, Institute of Microelectronics, University of Macau, Macau, China. and Faculty of Science and Technology - ECE, University of Macau, Macau, China
| | - Yanwei Jia
- State-Key Laboratory of Analog and Mixed-Signal VLSI, Institute of Microelectronics, University of Macau, Macau, China. and Faculty of Science and Technology - ECE, University of Macau, Macau, China and Faculty of Health Sciences, University of Macau, Macau, China
| | - Pui-In Mak
- State-Key Laboratory of Analog and Mixed-Signal VLSI, Institute of Microelectronics, University of Macau, Macau, China. and Faculty of Science and Technology - ECE, University of Macau, Macau, China
| | - Rui P Martins
- State-Key Laboratory of Analog and Mixed-Signal VLSI, Institute of Microelectronics, University of Macau, Macau, China. and Faculty of Science and Technology - ECE, University of Macau, Macau, China and On Leave from Instituto Superior Técnico, Universidade de Lisboa, Portugal
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22
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Wu H, Qian C, Wu C, Wang Z, Wang D, Ye Z, Ping J, Wu J, Ji F. End-point dual specific detection of nucleic acids using CRISPR/Cas12a based portable biosensor. Biosens Bioelectron 2020; 157:112153. [PMID: 32250930 DOI: 10.1016/j.bios.2020.112153] [Citation(s) in RCA: 70] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2019] [Revised: 02/27/2020] [Accepted: 03/12/2020] [Indexed: 12/26/2022]
Abstract
A CRISPR/Cas12a based portable biosensor (Cas12a-PB) was developed to simultaneously visually detect CaMV35S promoter and Lectin gene from genetically modified (GM) soybean powders (Roundup Ready@). The Cas12a-PB, mainly made of polymethylmethacrylate (PMMA) and PMMA tape, has a connection structure, three channels and three detection chambers. The CRISPR/Cas12a detection reagents were preloaded in detection chambers and the reaction tube was connected to the connection structure by screw threads. After amplification, the amplicons were gone into three detection chambers by swinging the Cas12a-PB to conduct dual detection. Positive samples would produce green fluorescence while negative samples were black under the irradiation of 490 nm LED light. In this study, the Cas12a-PB successively combined with ordinary PCR, rapid PCR and loop-mediated isothermal amplification (LAMP) to achieve dual detection, which made detection process more convenient and portable. As low as 0.1% transgenic ingredients in soybean powders could be detected and the specificity of Cas12a-PB was confirmed with GM maize powders (MON810, GA21), GM soybean powders (DP305423), non-GM peanut and rice as targets. In the end, an amplification chamber combining with Cas12a-PB on a PMMA chip was further designed to eliminate the use of reaction tube and mineral oil, which made operation simpler. The established Cas12a-PB would provide a new reliable solution for multiple targets detection in clinic diagnostics, food safety, etc.
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Affiliation(s)
- Hui Wu
- College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
| | - Cheng Qian
- College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
| | - Cui Wu
- College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
| | - Zhen Wang
- College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
| | - Dacheng Wang
- College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
| | - Zunzhong Ye
- College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
| | - Jianfeng Ping
- College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
| | - Jian Wu
- College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China; Key Laboratory of on Site Processing Equipment for Agricultural Products, Ministry of Agriculture, Hangzhou, 310058, China.
| | - Feng Ji
- The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China.
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23
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Sidstedt M, Rådström P, Hedman J. PCR inhibition in qPCR, dPCR and MPS-mechanisms and solutions. Anal Bioanal Chem 2020; 412:2009-2023. [PMID: 32052066 PMCID: PMC7072044 DOI: 10.1007/s00216-020-02490-2] [Citation(s) in RCA: 147] [Impact Index Per Article: 29.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Revised: 01/29/2020] [Accepted: 02/04/2020] [Indexed: 12/24/2022]
Abstract
DNA analysis has seen an incredible development in terms of instrumentation, assays and applications over the last years. Massively parallel sequencing (MPS) and digital PCR are now broadly applied in research and diagnostics, and quantitative PCR is used for more and more practises. All these techniques are based on in vitro DNA polymerization and fluorescence measurements. A major limitation for successful analysis is the various sample-related substances that interfere with the analysis, i.e. PCR inhibitors. PCR inhibition affects library preparation in MPS analysis and skews quantification in qPCR, and some inhibitors have been found to quench the fluorescence of the applied fluorophores. Here, we provide a deeper understanding of mechanisms of specific PCR inhibitors and how these impact specific analytical techniques. This background knowledge is necessary in order to take full advantage of modern DNA analysis techniques, specifically for analysis of samples with low amounts of template and high amounts of background material. The classical solution to handle PCR inhibition is to purify or dilute DNA extracts, which leads to DNA loss. Applying inhibitor-tolerant DNA polymerases, either single enzymes or blends, provides a more straightforward and powerful solution. This review includes mechanisms of specific PCR inhibitors as well as solutions to the inhibition problem in relation to cutting-edge DNA analysis.
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Affiliation(s)
- Maja Sidstedt
- Swedish National Forensic Centre, Swedish Police Authority, 581 94, Linköping, Sweden
| | - Peter Rådström
- Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, 221 00, Lund, Sweden
| | - Johannes Hedman
- Swedish National Forensic Centre, Swedish Police Authority, 581 94, Linköping, Sweden.
- Applied Microbiology, Department of Chemistry, Lund University, P.O. Box 124, 221 00, Lund, Sweden.
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24
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Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis. CANADIAN JOURNAL OF INFECTIOUS DISEASES & MEDICAL MICROBIOLOGY 2020; 2020:2697230. [PMID: 32184908 PMCID: PMC7061119 DOI: 10.1155/2020/2697230] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/16/2019] [Revised: 11/22/2019] [Accepted: 12/13/2019] [Indexed: 01/23/2023]
Abstract
Background and Objective. Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis are primary respiratory bacterial pathogens contributing to morbidity and mortality in developing countries. This study evaluated the diagnostic performance of multiplex real-time PCR with fluorescence melting curve analysis (MCA) assay, which was used to detect eight respiratory bacterial pathogens simultaneously. Methods A total of 157 sputum specimens were examined by multiplex real-time with fluorescence MCA, and the results were compared with the conventional culture method. Results Multiplex real-time PCR with fluorescence MCA specifically detected and differentiated eight respiratory bacterial pathogens by different melting curve peaks for each amplification product within 2 hours and exhibited high repeatability. The limit of detection ranged from 64 to 102 CFU/mL in the multiplex PCR system. Multiplex real-time PCR with fluorescence MCA showed a sensitivity greater than 80% and a 100% specificity for each pathogen. The kappa correlation of eight bacteria ranged from 0.89 to 1.00, and the coefficient of variation ranged from 0.05% to 0.80%. Conclusions Multiplex real-time PCR with fluorescence MCA assay is a sensitive, specific, high-throughput, and cost-effective method to detect multiple bacterial pathogens simultaneously.
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25
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Hagström L, Marques ALP, Nitz N, Hecht MM. The use of qPCR in human Chagas disease: a systematic review. Expert Rev Mol Diagn 2019; 19:875-894. [DOI: 10.1080/14737159.2019.1659729] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Affiliation(s)
- Luciana Hagström
- Laboratório Interdisciplinar de Biociências, Faculdade de Medicina, Universidade de Brasília, Campus Darcy Ribeiro, Brasília, Brazil
| | - Ana Luisa Pereira Marques
- Laboratório Interdisciplinar de Biociências, Faculdade de Medicina, Universidade de Brasília, Campus Darcy Ribeiro, Brasília, Brazil
| | - Nadjar Nitz
- Laboratório Interdisciplinar de Biociências, Faculdade de Medicina, Universidade de Brasília, Campus Darcy Ribeiro, Brasília, Brazil
| | - Mariana Machado Hecht
- Laboratório Interdisciplinar de Biociências, Faculdade de Medicina, Universidade de Brasília, Campus Darcy Ribeiro, Brasília, Brazil
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26
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Wang M, Hang J, Abuzeid AMI, Huang Y, Fu Y, Yan X, Zhang P, Huo C, Liu Y, Ran R, Sun Y, Li G. Development of multi-ARMS-qPCR method for detection of hookworms from cats and dogs. Parasitol Int 2019; 73:101974. [PMID: 31421266 DOI: 10.1016/j.parint.2019.101974] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2018] [Revised: 08/06/2019] [Accepted: 08/13/2019] [Indexed: 11/17/2022]
Abstract
Hookworms are blood-sucking nematodes that infect dogs, cats, and humans, causing iron-deficiency anemia, abdominal pain, diarrhea, and skin inflammation. Amplification refractory mutation system (ARMS) is a modified technology based on allele-specific PCR, which is widely used in mutation detection and genotyping. However, no data about ARMS application in hookworm detection. This study aims to establish a multi-ARMS-qPCR method for the detection of three hookworm species from dogs and cats. A universal forward primer and three specific primers (ARMS-Cey, ARMS-Can, and ARMS-Tub) were designed based on the three ITS SNPs (ITS250, ITS78 and ITS153) of Ancylostoma ceylanicum, A. caninum, and A. tubaeforme, respectively. The results showed that the three designed ARMS primers generated specific melting curves for the three hookworms' standard plasmids. The melting temperature (Tm) values were 88.40 °C (A. ceylanicum), 83.15 °C (A. caninum), and 85.65 °C (A. tubaeforme), with good reproducibility of intra- and inter-assay. No amplification was observed with other intestinal parasites. The limit of detection using the established technique was 1, 2, and 104 egg per gram feces (EPG) for A. caninum, A. tubaeforme and A. ceylanicum, respectively. Using multi-ARMS-qPCR assay, 17 out of 50 fecal samples were positive for hookworms, including ten single and seven mixed infections, and single infections were quantified. In conclusion, the used multi-ARMS-qPCR method has the advantages of high efficiency, sensitivity, specificity, and quantitative analysis and can be used for the clinical detection, epidemiological investigation, and zoonotic risk assessment of canine and feline hookworms.
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Affiliation(s)
- Mingwei Wang
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Jianxiong Hang
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Asmaa M I Abuzeid
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Yue Huang
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Yeqi Fu
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Xinxin Yan
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Pan Zhang
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Chenyang Huo
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Yunqiu Liu
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Rongkun Ran
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Yongxiang Sun
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China
| | - Guoqing Li
- Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510542, China.
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27
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Zhou M, Teng X, Li Y, Deng R, Li J. Cascade Transcription Amplification of RNA Aptamer for Ultrasensitive MicroRNA Detection. Anal Chem 2019; 91:5295-5302. [PMID: 30912425 DOI: 10.1021/acs.analchem.9b00124] [Citation(s) in RCA: 72] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
MicroRNAs (miRNAs) play a critical role in multifarious biological processes and being deemed to be important biomarkers for clinical cancer diagnosis, prognosis, and therapy. Thus, assays for sensitive and accurate quantification of miRNAs are highly demanded. Herein, we have constructed a RNA aptamer involved cascade transcription amplification method (termed RACTA), enabling label-free, ultrasensitive, and specific detection of miRNA. Target miRNA-initiated strand-displacement amplification would allow for the production of plenty of ssDNA that triggers the subsequent transcriptional amplification of spinach RNA aptamers. Consequently, transcribed tremendous spinach aptamers activated fluorophore DFHBI (( Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1 H-imidazol-5(4 H)-one) for miRNA quantitative analysis. RACTA outperforms conventional strand displacement amplification (SDA) at both background and amplification rate due to the light-up mechanism of DFHBI dye-Spinach aptamer and cascade signal amplification of RACTA. Thus, the signal-to-noise ratio of RACTA was increased by about 20-fold compared to that of SDA. This RACTA assay could confer a highly sensitive detection of miRNA with a detection limit of 5.12 × 10-18 M and excellent specificity enabling differentiation between miRNAs and homologous families. Besides, this assay has been successfully demonstrated for quantification of miRNAs in different cell lines. Therefore, the proposed method holds great potential for miRNA biomarker based early diagnosis and prognosis monitoring.
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Affiliation(s)
- Mengxi Zhou
- Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology , Tsinghua University , Beijing 100084 , China
| | - Xucong Teng
- Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology , Tsinghua University , Beijing 100084 , China
| | - Yue Li
- Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology , Tsinghua University , Beijing 100084 , China
| | - Ruijie Deng
- Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology , Tsinghua University , Beijing 100084 , China
| | - Jinghong Li
- Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology , Tsinghua University , Beijing 100084 , China
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28
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Zhang S, Assaf KI, Huang C, Hennig A, Nau WM. Ratiometric DNA sensing with a host-guest FRET pair. Chem Commun (Camb) 2019; 55:671-674. [PMID: 30565597 DOI: 10.1039/c8cc09126a] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
A supramolecular host-guest FRET pair based on a carboxyfluorescein-labelled cucurbit[7]uril (CB7-CF, as acceptor) and the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI, as donor) is developed for sensing of DNA. In comparison to the commercial DNA staining dye SYBR Green I, the new chemosensing ensemble offers dual-emission signals, which allows a linear ratiometric response over a wide concentration range.
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Affiliation(s)
- Shuai Zhang
- Department of Life Sciences and Chemistry, Jacobs University Bremen, Campus Ring 1, 28759, Bremen, Germany.
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29
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Ma Q, Gao Z. A simple and ultrasensitive fluorescence assay for single-nucleotide polymorphism. Anal Bioanal Chem 2018; 410:3093-3100. [PMID: 29644378 DOI: 10.1007/s00216-018-0874-4] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2017] [Revised: 12/11/2017] [Accepted: 01/12/2018] [Indexed: 11/30/2022]
Abstract
In this report, a simple, label-free and highly efficient nucleic acid amplification technique is developed for ultrasensitive detection of single-nucleotide polymorphism (SNP). Briefly, a designed padlock probe is first circularized by a DNA ligase when it perfectly complements to a mutant gene. Then, the mutant gene functions as a primer to initiate branched rolling circle amplification reaction (BRCA), generating a large number of branched DNA strands and a lot of pyrophosphate molecules which is equivalent to the number of nucleotides consumed. With the addition of a terpyridine-Zn(II) complex, pyrophosphate molecules can be sensitively detected owing to the formation of a fluorescent terpyridine-Zn(II)-pyrophosphate complex. The fluorescence intensity is directly associated with the content of the mutant gene in a sample solution. On the other hand, the circulation of the padlock probe is prohibited when it hybridizes with the wild-type gene. In this assay, the accumulative nature of the BRCA process produces a detection limit of 0.1 pM and an excellent selectivity factor of 1000 toward SNP. As little as 0.1% mutant in the wild-type gene can be successfully detected. The simple procedure, high sensitivity, and high selectivity of this assay offer a potentially viable alternative for routine SNP analysis. Graphical abstract A simple and label-free fluorescence assay for SNP detection by coupling BRCA with selective fluorescence detection of pyrophosphate using the terpyridine-Zn(II) complex.
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Affiliation(s)
- Qian Ma
- Department of Chemistry, National University of Singapore, 3 Science Drive 2, Singapore, 117543, Singapore
| | - Zhiqiang Gao
- Department of Chemistry, National University of Singapore, 3 Science Drive 2, Singapore, 117543, Singapore.
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30
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Wang ZY, Wang LJ, Zhang Q, Tang B, Zhang CY. Single quantum dot-based nanosensor for sensitive detection of 5-methylcytosine at both CpG and non-CpG sites. Chem Sci 2018; 9:1330-1338. [PMID: 29675180 PMCID: PMC5887231 DOI: 10.1039/c7sc04813k] [Citation(s) in RCA: 55] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2017] [Accepted: 12/12/2017] [Indexed: 12/13/2022] Open
Abstract
DNA methylation is an important epigenetic modification in human genomes. Herein, we develop a single quantum dot (QD)-based nanosensor for sensitive detection of DNA methylation at both CpG and non-CpG sites using tricyclic ligation chain reaction (LCR)-mediated QD-based fluorescence resonance energy transfer (FRET). We design two sets of DNA probes (X and Y, X' and Y') for methylated DNA assay. In the presence of thermostable DNA ligase, probes X and Y may adjacently hybridize with the methylated DNA to obtain the ligated XY products which may function as the templates for probes X' and Y' to generate the X'Y' products. The resultant X'Y' products may in turn act as the templates to ligate probes X and Y for the generation of XY products, consequently inducing tricyclic LCR amplification under thermal denaturation conditions to generate a large number of XY products. The subsequent hybridization of XY products with the capture and reporter probes results in the formation of sandwich hybrids which may assemble on the 605QD surface to obtain 605QD-oligonucleotide-Cy5 nanostructures, inducing efficient FRET from the 605QD to Cy5 and the emission of Cy5. This nanosensor can detect DNA methylation at single 5-methylcytosine (5-mC) resolution with a detection limit of as low as 1.0 aM and a large dynamic range of 7 orders of magnitude. Moreover, this nanosensor can distinguish as low as a 0.01% methylation level, and it can detect DNA methylation in human lung cancer cells as well, holding great potential for accurate epigenetic evaluation and early cancer diagnosis.
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Affiliation(s)
- Zi-Yue Wang
- College of Chemistry, Chemical Engineering and Materials Science , Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong , Key Laboratory of Molecular and Nano Probes , Ministry of Education , Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China . ; ; ; Tel: +86-0531-86186033
| | - Li-Juan Wang
- College of Chemistry, Chemical Engineering and Materials Science , Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong , Key Laboratory of Molecular and Nano Probes , Ministry of Education , Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China . ; ; ; Tel: +86-0531-86186033
| | - Qianyi Zhang
- Nantou High School Shenzhen , Shenzhen , 518052 , China
| | - Bo Tang
- College of Chemistry, Chemical Engineering and Materials Science , Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong , Key Laboratory of Molecular and Nano Probes , Ministry of Education , Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China . ; ; ; Tel: +86-0531-86186033
| | - Chun-Yang Zhang
- College of Chemistry, Chemical Engineering and Materials Science , Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong , Key Laboratory of Molecular and Nano Probes , Ministry of Education , Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China . ; ; ; Tel: +86-0531-86186033
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31
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Lubis H, Salihah NT, Norizan NA, Hossain MM, Ahmed MU. Fast and Sensitive Real-time PCR-based Detection of Porcine DNA in Food Samples by Using EvaGreen Dye. FOOD SCIENCE AND TECHNOLOGY RESEARCH 2018. [DOI: 10.3136/fstr.24.803] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Affiliation(s)
- Hamadah Lubis
- Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam
| | - Nur Thaqifah Salihah
- Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam
| | - Nur Aqirah Norizan
- Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam
| | | | - Minhaz Uddin Ahmed
- Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam
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32
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Kim HY, Li T, Jung C, Fu R, Cho DY, Park KS, Park HG. Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs. RSC Adv 2018; 8:37391-37395. [PMID: 35557795 PMCID: PMC9089284 DOI: 10.1039/c8ra06675b] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2018] [Accepted: 10/28/2018] [Indexed: 11/21/2022] Open
Abstract
We herein describe a novel quantitative PCR (qPCR) method, which operates in both signal-off and on manners, by utilizing a unique property of fluorescent nucleobase analogs. The first, signal-off method is developed by designing the primers to contain pyrrolo-dC (PdC), one of the most common fluorescent nucleobase analogs. The specially designed single-stranded primer is extended to form double-stranded DNA during PCR and the fluorescence signal from the PdCs incorporated in the primer is accordingly reduced due to its conformation-dependent fluorescence properties. In addition, the second, signal-on method is devised by designing the primers to contain 5′-overhang sequences complementary to the PdC-incorporated DNA probes. At the initial phase, the PdC-incorporated DNA probes are hybridized to the 5′-overhang sequences of the primer, exhibiting the significantly quenched fluorescence signal, but are detached by either hydrolysis or strand displacement reaction during PCR, leading to the highly enhanced fluorescence signal. This method is more advanced than the first one since it produces signal-on fluorescence response and permits the use of a single PdC-incorporated DNA probe for the detection of multiple target nucleic acids, remarkably decreasing the assay cost. With these novel qPCR methods, we successfully quantified target nucleic acids derived from sexually transmitted disease (STD) pathogens with high accuracy. Importantly, the proposed strategies overcome the major drawbacks in the current SYBR Green and TaqMan probe-based qPCR methods such as low specificity and high assay cost. A novel quantitative PCR (qPCR) method was developed by utilizing a unique property of fluorescent nucleobase analogs (PdCs).![]()
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Affiliation(s)
- Hyo Yong Kim
- Department of Chemical and Biomolecular Engineering (BK21 Program)
- KAIST
- Daejeon 305-701
- Republic of Korea
| | - Taihua Li
- College of Biology and the Environment
- Co-Innovation Centre for Sustainable Forestry in Southern China
- Nanjing Forestry University
- Nanjing
- China
| | - Cheulhee Jung
- Department of Chemical and Biomolecular Engineering (BK21 Program)
- KAIST
- Daejeon 305-701
- Republic of Korea
| | - Rongzhan Fu
- Department of Chemical and Biomolecular Engineering (BK21 Program)
- KAIST
- Daejeon 305-701
- Republic of Korea
| | - Dae-Yeon Cho
- Labgenomics Clinical Research Institute
- Labgenomics Co. Ltd
- Yong-In
- Republic of Korea
| | - Ki Soo Park
- Department of Biological Engineering
- College of Engineering
- Konkuk University
- Seoul 05029
- Republic of Korea
| | - Hyun Gyu Park
- Department of Chemical and Biomolecular Engineering (BK21 Program)
- KAIST
- Daejeon 305-701
- Republic of Korea
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33
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Suseela YV, Narayanaswamy N, Pratihar S, Govindaraju T. Far-red fluorescent probes for canonical and non-canonical nucleic acid structures: current progress and future implications. Chem Soc Rev 2018; 47:1098-1131. [DOI: 10.1039/c7cs00774d] [Citation(s) in RCA: 126] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Our review presents the recent progress on far-red fluorescent probes of canonical and non-canonical nucleic acid (NA) structures, critically discusses the design principles, applications, limitations and outline the future prospects of developing newer probes with target-specificity for different NA structures.
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Affiliation(s)
- Y. V. Suseela
- Bioorganic Chemistry Laboratory
- New Chemistry Unit
- Jawaharlal Nehru Centre for Advanced Scientific Research
- Bengaluru 560064
- India
| | - Nagarjun Narayanaswamy
- Bioorganic Chemistry Laboratory
- New Chemistry Unit
- Jawaharlal Nehru Centre for Advanced Scientific Research
- Bengaluru 560064
- India
| | - Sumon Pratihar
- Bioorganic Chemistry Laboratory
- New Chemistry Unit
- Jawaharlal Nehru Centre for Advanced Scientific Research
- Bengaluru 560064
- India
| | - Thimmaiah Govindaraju
- Bioorganic Chemistry Laboratory
- New Chemistry Unit
- Jawaharlal Nehru Centre for Advanced Scientific Research
- Bengaluru 560064
- India
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34
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Sensitive and specific detection of microRNAs based on two-stage amplification reaction using molecular beacons as turn-on probes. Talanta 2017; 179:685-692. [PMID: 29310294 DOI: 10.1016/j.talanta.2017.11.068] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2017] [Revised: 11/16/2017] [Accepted: 11/28/2017] [Indexed: 12/13/2022]
Abstract
In this study, a rapid, sensitive, and specific assay for detecting miRNAs was developed based on a two-stage amplification reaction (TSAR) using molecular beacons (MBs) as turn-on probes. In the TSAR, different miRNAs can be converted to the same reporter oligonucleotides (Y), which can hybridize with the same MB. Therefore, in combination with specific templates, this method can be applied to multiplex miRNA detection by simply using the same MB. The loop region of the MB was screened by computer simulation methods. In particular, to improve the specificity of the MB in real sample analysis, the maximum similarity of the MB loop region to the human genome and human transcriptome is less than 70%. Two MBs were designed in this study. MB I, with nine flanking base pairs in its stem region, was used for real-time monitoring of the production of Y during the TSAR. MB II, with five flanking base pairs in its stem region, was used to detect the production of Y at the end of the TSAR. This assay exhibited high sensitivity with a limit of detection of 2.0 × 10-16M and 6.7 × 10-16M using MB I and MB II as turn-on probes, respectively. In addition, this assay can clearly discriminate single base differences in miRNA sequences, and the TSAR can be completed under isothermal conditions. Accordingly, the isothermal reaction conditions and simple fluorescence measurement can greatly contribute to the development of a fast point-of-care detection system.
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35
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A novel quantitative PCR mediated by high-fidelity DNA polymerase. Sci Rep 2017; 7:10365. [PMID: 28871131 PMCID: PMC5583327 DOI: 10.1038/s41598-017-10782-4] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2017] [Accepted: 08/14/2017] [Indexed: 01/19/2023] Open
Abstract
The biggest challenge for accurate diagnosis of viral infectious disease is the high genetic variability of involved viruses, which affects amplification efficiency and results in low sensitivity and narrow spectrum. Here, we developed a new simple qPCR mediated by high-fidelity (HF) DNA polymerase. The new method utilizes an HFman probe and one primer. Fluorescent signal was generated from the 3'-5' hydrolysis of HFman probe by HF DNA polymerase before elongation initiation. Mismatches between probe/primer and template have less influence on the amplification efficiency of the new method. The new qPCR exhibited higher sensitivity and better adaptability to sequence variable templates than the conventional TaqMan probe based-qPCR in quantification of HIV-1 viral load. Further comparison with COBAS TaqMan HIV-1 Test (v2.0) showed a good correlation coefficient (R2 = 0.79) between both methods in quantification of HIV-1 viral load among 21 clinical samples. The characteristics of tolerance to variable templates and one probe-one primer system imply that the probe/primer design for the new method will be easier and more flexible than the conventional method for highly heterogeneous viruses. Therefore, the HF DNA polymerase-mediated qPCR method is a simple, sensitive and promising approach for the development of diagnostics for viral infectious diseases.
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36
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Li Y, Liu N, Liu H, Wang Y, Hao Y, Ma X, Li X, Huo Y, Lu J, Tang S, Wang C, Zhang Y, Gao Z. A novel label-free fluorescence assay for one-step sensitive detection of Hg 2+ in environmental drinking water samples. Sci Rep 2017; 7:45974. [PMID: 28378768 PMCID: PMC5380999 DOI: 10.1038/srep45974] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2016] [Accepted: 03/07/2017] [Indexed: 01/11/2023] Open
Abstract
A novel label-free fluorescence assay for detection of Hg2+ was developed based on the Hg2+-binding single-stranded DNA (ssDNA) and SYBR Green I (SG I). Differences from other assays, the designed rich-thymine (T) ssDNA probe without fluorescent labelling can be rapidly formed a T-Hg2+-T complex and folded into a stable hairpin structure in the presence of Hg2+ in environmental drinking water samples by facilitating fluorescence increase through intercalating with SG I in one-step. In the assay, the fluorescence signal can be directly obtained without additional incubation within 1 min. The dynamic quantitative working ranges was 5–1000 nM, the determination coefficients were satisfied by optimization of the reaction conditions. The lowest detection limit of Hg2+ was 3 nM which is well below the standard of U.S. Environmental Protection Agency. This method was highly specific for detecting of Hg2+ without being affected by other possible interfering ions from different background compositions of water samples. The recoveries of Hg2+ spiked in these samples were 95.05–103.51%. The proposed method is more viable, low-costing and simple for operation in field detection than the other methods with great potentials, such as emergency disposal, environmental monitoring, surveillance and supporting of ecological risk assessment and management.
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Affiliation(s)
- Ya Li
- School of Public Health, Lanzhou University, Lanzhou 73000, P. R. China.,Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Health and Environmental Medicine, Tianjin, 300050, P. R. China
| | - Nan Liu
- School of Public Health, Lanzhou University, Lanzhou 73000, P. R. China.,Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Health and Environmental Medicine, Tianjin, 300050, P. R. China.,School of Public Health, State Ministry of Education, Sun Yat-sen University, Guangzhou, Guangdong, 510080, P. R. China.,Department of Nutrition and Food Hygiene, College of Public Health, Zhengzhou University, Zhengzhou, 450001, P. R. China
| | - Hui Liu
- School of Public Health, Lanzhou University, Lanzhou 73000, P. R. China
| | - Yu Wang
- School of Public Health, Lanzhou University, Lanzhou 73000, P. R. China
| | - Yuwei Hao
- School of Public Health, Lanzhou University, Lanzhou 73000, P. R. China
| | - Xinhua Ma
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Health and Environmental Medicine, Tianjin, 300050, P. R. China
| | - Xiaoli Li
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Health and Environmental Medicine, Tianjin, 300050, P. R. China
| | - Yapeng Huo
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Health and Environmental Medicine, Tianjin, 300050, P. R. China
| | - Jiahai Lu
- School of Public Health, State Ministry of Education, Sun Yat-sen University, Guangzhou, Guangdong, 510080, P. R. China
| | - Shuge Tang
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Health and Environmental Medicine, Tianjin, 300050, P. R. China.,Department of Nutrition and Food Hygiene, College of Public Health, Zhengzhou University, Zhengzhou, 450001, P. R. China
| | - Caiqin Wang
- School of Public Health, Lanzhou University, Lanzhou 73000, P. R. China.,Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Health and Environmental Medicine, Tianjin, 300050, P. R. China
| | - Yinhong Zhang
- School of Public Health, Lanzhou University, Lanzhou 73000, P. R. China
| | - Zhixian Gao
- Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Health and Environmental Medicine, Tianjin, 300050, P. R. China
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37
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Liu H, Tian T, Zhang Y, Ding L, Yu J, Yan M. Sensitive and rapid detection of microRNAs using hairpin probes-mediated exponential isothermal amplification. Biosens Bioelectron 2017; 89:710-714. [DOI: 10.1016/j.bios.2016.10.099] [Citation(s) in RCA: 54] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2016] [Revised: 10/09/2016] [Accepted: 10/21/2016] [Indexed: 10/20/2022]
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38
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Park J, Song M, Jang W, Chae H, Lee GD, Kim K, Park H, Kim M, Kim Y. Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations. Clin Chim Acta 2016; 465:82-90. [PMID: 27939919 DOI: 10.1016/j.cca.2016.12.002] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2016] [Revised: 11/30/2016] [Accepted: 12/02/2016] [Indexed: 01/26/2023]
Abstract
BACKGROUND We developed and evaluated the feasibility of peptide nucleic acid (PNA)-based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). METHODS We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &CALR p.Leu367fs*46 (set A) and MPL W515L/K &CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger sequencing in 57 BCR-ABL1-negative MPNs. RESULTS The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed. Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8). Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by this method owing to invading of probe binding site. CONCLUSIONS PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with >10% mutant allele at the time of initial diagnosis.
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Affiliation(s)
- Joonhong Park
- Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Minsik Song
- SeaSun Biomaterials, Daejeon, Republic of Korea
| | - Woori Jang
- Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Hyojin Chae
- Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Gun Dong Lee
- Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | | | | | - Myungshin Kim
- Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
| | - Yonggoo Kim
- Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
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Abstract
Quantitative real-time polymerase chain reaction is a technique for simultaneous amplification and product quantification of a target DNA as the process takes place in real time in a "closed-tube" system. Although this technique can provide an absolute quantification of the initial template copy number, quantification relative to a control sample or second sequence is typically adequate. The quantification process employs melting curve analysis and/or fluorescent detection systems and can provide amplification and genotyping in a relatively short time. Here we describe the properties and uses of various fluorescent detection systems used for quantification.
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Affiliation(s)
- Charanjeet Singh
- Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Sinchita Roy-Chowdhuri
- Department of Pathology, Cytopathology and Molecular Pathology, MD Anderson Cancer Center, The University ofTexas, Houston, TX, USA.
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Simintiras CA, Fröhlich T, Sathyapalan T, Arnold GJ, Ulbrich SE, Leese HJ, Sturmey RGS. Modelling oviduct fluid formation in vitro. Reproduction 2016; 153:REP-15-0508. [PMID: 27738189 DOI: 10.1530/rep-15-0508] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2015] [Accepted: 10/13/2016] [Indexed: 02/28/2024]
Abstract
Oviduct fluid is the microenvironment that supports early reproductive processes including fertilisation, embryo cleavage, and genome activation. However, the composition and regulation of this critical environment remains rather poorly defined. This study uses an in vitro preparation of the bovine oviduct epithelium, to investigate the formation and composition of in vitro derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct specific glycoprotein 1 in ivDOF and show that the amino acid and carbohydrate content resembles that of previously reported in vivo data. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes, and the corresponding flux of amino acids within ivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully the in vivo environment and impacts on ivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on the in vitro oviduct preparation evaluated. These experiments help clarify the dynamic function of the oviduct in vitro and suggest a number of future research avenues, such as investigating epithelial-fibroblast interactions, probing the molecular aetiologies of subfertility, and optimising embryo culture media.
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Affiliation(s)
- Constantine A Simintiras
- C Simintiras, Centre for Cardiovascular and Metabolic Research (CCMR), Hull York Medical School (HYMS), Kingston upon Hull, United Kingdom of Great Britain and Northern Ireland
| | - Thomas Fröhlich
- T Fröhlich, Laboratory for Functional Genome Analysis (LAFUGA), LMU Munich, Munich, Germany
| | - Thozhukat Sathyapalan
- T Sathyapalan, Michael White Centre for Diabetes and Endocrinology, Hull York Medical School (HYMS), Kingston upon Hull, Hu32rw, United Kingdom of Great Britain and Northern Ireland
| | - Georg J Arnold
- G Arnold, Laboratory for Functional Genome Analysis (LAFUGA), LMU Munich, Munich, Germany
| | - Susanne E Ulbrich
- S Ulbrich, Animal Physiology, ETH Zurich, Institute of Agricultural Sciences, Zurich, Switzerland
| | - Henry J Leese
- H Leese, Centre for Cardiovascular and Metabolic Research (CCMR), Hull York Medical School (HYMS), Kingston upon Hull, United Kingdom of Great Britain and Northern Ireland
| | - Roger G S Sturmey
- R Sturmey, Centre for Cardiovascular and Metabolic Research (CCMR), Hull York Medical School (HYMS), Kingston upon Hull, United Kingdom of Great Britain and Northern Ireland
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41
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Aguayo-Patrón S, Beltrán-Sauceda L, Calderón de la Barca AM. A population-wide applicable HLA-DQ2 and DQ8 genotyping using DNA from dried blood spots and duplex allele-specific qPCR amplification. Scandinavian Journal of Clinical and Laboratory Investigation 2016; 76:581-587. [PMID: 27670799 DOI: 10.1080/00365513.2016.1230773] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Genotyping of HLA-DQ2 and DQ8 haplotypes is important for diagnosis or for screening of early risk detection of celiac disease or type 1 diabetes. Usually, venous blood DNA extraction and expensive and time consuming amplification are used, that hinder population-wide studies. We assayed a friendly HLA-DQ2 and DQ8 genotyping procedure using a combination of DNA from dried blood spot (DBS) and duplex allele-specific qPCR amplification using SYBR Green. DNA was extracted using home-made buffers and compared to an extraction commercial kit. Duplex reactions by qPCR were designed using each Tm allele amplicon for reference samples (positive HLA-DQ2 or DQ8) with allele-specific primers. DBS samples from 558 children (7.99 ± 2.47 y) were collected. The DNA final yield obtained by the home-made extractive procedure was higher than from the commercial kit (1.11 ± 0.56 vs 0.23 ± 0.14 μg), while the quality was similar for both DNA samples. There was concordance in the amplification profiles for DNA samples obtained with both methods. All of four alleles from DQ2 and DQ8 haplotypes were accurately identified in duplex reactions. By using DBS samples and DNA extraction home-made procedure, the costs were reduced by 60%. The whole procedure is cost-effective for HLA-DQ2 and DQ8 genotyping.
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Affiliation(s)
- Sandra Aguayo-Patrón
- a Coordinación de Nutrición , Centro de Investigación en Alimentación y Desarrollo , Hermosillo , Sonora , México
| | - Lizbeth Beltrán-Sauceda
- a Coordinación de Nutrición , Centro de Investigación en Alimentación y Desarrollo , Hermosillo , Sonora , México
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42
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He P, Zhu G, Luo J, Wang H, Yan Y, Chen L, Gao W, Chen Z. Development and Application of a One-Tube Multiplex Real-Time PCR with Melting Curve Analysis for Simultaneous Detection of Five Foodborne Pathogens in Food Samples. J Food Saf 2016. [DOI: 10.1111/jfs.12297] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Affiliation(s)
- Peiyan He
- Jiaxing Center for Disease Control and Prevention; Jiaxing 314050 P.R. China
| | - Guoying Zhu
- Jiaxing Center for Disease Control and Prevention; Jiaxing 314050 P.R. China
| | - Jianyong Luo
- Jiaxing Center for Disease Control and Prevention; Jiaxing 314050 P.R. China
| | - Henghui Wang
- Jiaxing Center for Disease Control and Prevention; Jiaxing 314050 P.R. China
| | - Yong Yan
- Jiaxing Center for Disease Control and Prevention; Jiaxing 314050 P.R. China
| | - Lixia Chen
- Jiaxing Center for Disease Control and Prevention; Jiaxing 314050 P.R. China
| | - Wenjie Gao
- Jiaxing Center for Disease Control and Prevention; Jiaxing 314050 P.R. China
| | - Zhongwen Chen
- Jiaxing Center for Disease Control and Prevention; Jiaxing 314050 P.R. China
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43
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Irshad M, Gupta P, Mankotia DS, Ansari MA. Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review. World J Gastroenterol 2016; 22:4824-4834. [PMID: 27239109 PMCID: PMC4873875 DOI: 10.3748/wjg.v22.i20.4824] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/09/2016] [Revised: 03/09/2016] [Accepted: 03/30/2016] [Indexed: 02/06/2023] Open
Abstract
The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.
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MESH Headings
- DNA, Viral/blood
- DNA, Viral/genetics
- Hepatitis Viruses/classification
- Hepatitis Viruses/genetics
- Hepatitis Viruses/immunology
- Hepatitis, Viral, Human/blood
- Hepatitis, Viral, Human/diagnosis
- Hepatitis, Viral, Human/genetics
- Hepatitis, Viral, Human/immunology
- Humans
- Multiplex Polymerase Chain Reaction
- Predictive Value of Tests
- Reproducibility of Results
- Serogroup
- Serologic Tests/methods
- Serotyping
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44
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Barthelmes J, Tafferner N, Kurz J, de Bruin N, Parnham MJ, Geisslinger G, Schiffmann S. Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues. J Vis Exp 2016. [PMID: 27214391 DOI: 10.3791/53933] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1β, IL-6, IL-23, TNFα, IFNγ) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.
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Affiliation(s)
- Julia Barthelmes
- Institute of Clinical Pharmacology, Goethe University Hospital Frankfurt
| | - Nadja Tafferner
- Project Group for Translational Medicine & Pharmacology, Fraunhofer IME
| | - Jennifer Kurz
- Project Group for Translational Medicine & Pharmacology, Fraunhofer IME
| | - Natasja de Bruin
- Project Group for Translational Medicine & Pharmacology, Fraunhofer IME
| | - Michael J Parnham
- Project Group for Translational Medicine & Pharmacology, Fraunhofer IME
| | - Gerd Geisslinger
- Institute of Clinical Pharmacology, Goethe University Hospital Frankfurt
| | - Susanne Schiffmann
- Project Group for Translational Medicine & Pharmacology, Fraunhofer IME;
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Salihah NT, Hossain MM, Lubis H, Ahmed MU. Trends and advances in food analysis by real-time polymerase chain reaction. Journal of Food Science and Technology 2016; 53:2196-209. [PMID: 27407185 PMCID: PMC4921084 DOI: 10.1007/s13197-016-2205-0] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Revised: 03/06/2016] [Accepted: 03/14/2016] [Indexed: 02/05/2023]
Abstract
Analyses to ensure food safety and quality are more relevant now because of rapid changes in the quantity, diversity and mobility of food. Food-contamination must be determined to maintain health and up-hold laws, as well as for ethical and cultural concerns. Real-time polymerase chain reaction (RT-PCR), a rapid and inexpensive quantitative method to detect the presence of targeted DNA-segments in samples, helps in determining both accidental and intentional adulterations of foods by biological contaminants. This review presents recent developments in theory, techniques, and applications of RT-PCR in food analyses, RT-PCR addresses the limitations of traditional food analyses in terms of sensitivity, range of analytes, multiplexing ability, cost, time, and point-of-care applications. A range of targets, including species of plants or animals which are used as food ingredients, food-borne bacteria or viruses, genetically modified organisms, and allergens, even in highly processed foods can be identified by RT-PCR, even at very low concentrations. Microfluidic RT-PCR eliminates the separate sample-processing step to create opportunities for point-of-care analyses. We also cover the challenges related to using RT-PCR for food analyses, such as the need to further improve sample handling.
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Affiliation(s)
- Nur Thaqifah Salihah
- Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong, BE 1410 Brunei Darussalam
| | | | - Hamadah Lubis
- Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong, BE 1410 Brunei Darussalam
| | - Minhaz Uddin Ahmed
- Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong, BE 1410 Brunei Darussalam
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46
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Nagy A, Černíková L, Vitásková E, Křivda V, Dán Á, Dirbáková Z, Jiřincová H, Procházka B, Sedlák K, Havlíčková M. MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay. PLoS One 2016; 11:e0151204. [PMID: 27031831 PMCID: PMC4816343 DOI: 10.1371/journal.pone.0151204] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2015] [Accepted: 02/23/2016] [Indexed: 12/05/2022] Open
Abstract
In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.
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Affiliation(s)
- Alexander Nagy
- Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic
- National Reference Laboratory for Influenza, National Institute of Public Health, Prague, Czech Republic
- * E-mail:
| | - Lenka Černíková
- Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic
| | - Eliška Vitásková
- Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic
| | - Vlastimil Křivda
- Laboratory of Molecular Methods, State Veterinary Institute Prague, Prague, Czech Republic
- Department of Virology and Serology, State Veterinary Institute Prague, Prague, Czech Republic
| | - Ádám Dán
- National Food Chain Safety Office, Veterinary Diagnostic Directorate, Molecular Biology Laboratory, Budapest, Hungary
| | - Zuzana Dirbáková
- Department of Virology, State Veterinary Institute Zvolen, Zvolen, Slovak Republic
| | - Helena Jiřincová
- National Reference Laboratory for Influenza, National Institute of Public Health, Prague, Czech Republic
| | - Bohumír Procházka
- Department of Informatics and Biostatistics, National Institute of Public Health, Prague, Czech Republic
| | - Kamil Sedlák
- Department of Virology and Serology, State Veterinary Institute Prague, Prague, Czech Republic
| | - Martina Havlíčková
- National Reference Laboratory for Influenza, National Institute of Public Health, Prague, Czech Republic
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47
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Camacho Londoño J, Philipp SE. A reliable method for quantification of splice variants using RT-qPCR. BMC Mol Biol 2016; 17:8. [PMID: 26979160 PMCID: PMC4793508 DOI: 10.1186/s12867-016-0060-1] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2014] [Accepted: 03/03/2016] [Indexed: 12/19/2022] Open
Abstract
Background The majority of protein isoforms arise from alternative splicing of the encoding primary RNA transcripts. To understand the significance of single splicing events, reliable techniques are needed to determine their incidence. However, existing methods are labour-intensive, error-prone or of limited use. Results Here, we present an improved method to determine the relative incidence of transcripts that arise from alternative splicing at a single site. Splice variants were quantified within a single sample using one-step reverse transcription quantitative PCR. Amplification products obtained with variant specific primer pairs were compared to those obtained with primer pairs common to both variants. The identities of variant specific amplicons were simultaneously verified by melt curve analysis. Independent calculations of the relative incidence of each variant were performed. Since the relative incidences of variants have to add upto 100 %, the method provides an internal control to monitor experimental errors and uniform reverse transcription. The reliability of the method was tested using mixtures of cDNA templates as well as RNA samples from different sources. Conclusion The method described here, is easy to set up and does not need unrelated reference genes and time consuming, error-prone standard curves. It provides a reliable and precise technique to distinguish small differences of the relative incidence of two splice variants.
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Affiliation(s)
- Julia Camacho Londoño
- Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, 66421, Homburg, Germany
| | - Stephan E Philipp
- Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, 66421, Homburg, Germany.
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48
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A novel pentaplex real time (RT)- PCR high resolution melt curve assay for simultaneous detection of emetic and enterotoxin producing Bacillus cereus in food. Food Control 2016. [DOI: 10.1016/j.foodcont.2015.08.030] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
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49
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Price ER, Rott KH, Caviedes-Vidal E, Karasov WH. Claudin gene expression patterns do not associate with interspecific differences in paracellular nutrient absorption. Comp Biochem Physiol B Biochem Mol Biol 2016; 191:36-45. [DOI: 10.1016/j.cbpb.2015.09.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2015] [Revised: 09/08/2015] [Accepted: 09/09/2015] [Indexed: 11/27/2022]
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50
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Guo Y, Xu L, Hong S, Sun Q, Yao W, Pei R. Label-free DNA-based biosensors using structure-selective light-up dyes. Analyst 2016; 141:6481-6489. [DOI: 10.1039/c6an01958g] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Label-free biosensors (LFBs) have demonstrated great potential in cost-effective applications. This review collected the latest reported works which employed structure-selective nucleic acid dyes for the development of DNA-based LFBs.
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Affiliation(s)
- Yahui Guo
- State Key Laboratory of Food Science and Technology
- School of Food Science and Technology
- Jiangnan University
- Wuxi 214122
- China
| | - Lijun Xu
- Key Laboratory of Nano-Bio Interface
- Division of Nanobiomedicine
- Suzhou Institute of Nano-Tech and Nano-Bionics
- Chinese Academy of Sciences
- Suzhou 215123
| | - Shanni Hong
- Key Laboratory of Nano-Bio Interface
- Division of Nanobiomedicine
- Suzhou Institute of Nano-Tech and Nano-Bionics
- Chinese Academy of Sciences
- Suzhou 215123
| | - Qingqing Sun
- State Key Laboratory of Food Science and Technology
- School of Food Science and Technology
- Jiangnan University
- Wuxi 214122
- China
| | - Weirong Yao
- State Key Laboratory of Food Science and Technology
- School of Food Science and Technology
- Jiangnan University
- Wuxi 214122
- China
| | - Renjun Pei
- Key Laboratory of Nano-Bio Interface
- Division of Nanobiomedicine
- Suzhou Institute of Nano-Tech and Nano-Bionics
- Chinese Academy of Sciences
- Suzhou 215123
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