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1
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Lee EY, Lee M, Kim MG, Bae CE, Kim SH, Shin Y. Acid-activated bentonite for solid-phase nucleic acid extraction from various pathogenic samples. Anal Chim Acta 2025; 1352:343928. [PMID: 40210284 DOI: 10.1016/j.aca.2025.343928] [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: 12/13/2024] [Revised: 03/07/2025] [Accepted: 03/10/2025] [Indexed: 04/12/2025]
Abstract
BACKGROUND Despite significant advancements in nucleic acid testing technologies, current nucleic acid extraction methods are often limited by inefficiency, complexity, and a lack of versatility. To overcome these challenges, we have developed an innovative solid-phase extraction (SPE) method employing sulfuric acid-activated bentonite (SAB) for extracting nucleic acids from various sample types. RESULTS Activation with sulfuric acid expands the surface area of bentonite by 2.2 times, thereby enhancing its adsorption capacity and surface modification efficiency. To further improve extraction efficiency, we modified SAB through amine-functionalization using 3-aminopropyl(diethoxy)methylsilane (APDMS), resulting in the creation of APDMS-modified SAB (ASAB). This modification facilitates efficient nucleic acid binding via reversible interactions mediated by a homobifunctional imidoester (HI) reagent. Our ASAB-based SPE system offers a streamlined, universal protocol for isolating DNA, RNA, and miRNA from diverse samples, including clinical bodily fluids and culture media, in under 30 min. Moreover, the system effectively enriches low concentrations of negatively charged pathogens (down to 20 CFU/reaction) from large-volume samples (up to 50 mL) through a 30-min pre-enrichment step utilizing the positively charged ASAB-HI complex. Comparative testing with pooled human urine and plasma samples revealed up to a 3.95-fold increase in DNA recovery compared to commercial SPE kits. Additionally, the system demonstrated up to a 6.3-fold improvement in the isolation of unstable viral RNA from clinical nasopharyngeal swabs, as well as critical microRNA biomarkers. SIGNIFICANCE The versatility and high efficiency of nucleic acid recovery with our ASAB-based SPE system indicate its potential to revolutionize traditional SPE methods, positioning it as a universal nucleic acid extraction platform for molecular biology research.
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Affiliation(s)
- Eun Yeong Lee
- Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea
| | - Minju Lee
- Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea
| | - Myoung Gyu Kim
- Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea
| | - Chae Eun Bae
- Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea
| | - Sung-Han Kim
- Department of Infectious Disease, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
| | - Yong Shin
- Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Republic of Korea.
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Radwan RE, Darwish A, Elsaid AM, El-Kholy WM. Exploring the potential of IL-10 for risk assessment and early intervention in pediatric ALL. BMC Cancer 2024; 24:972. [PMID: 39118076 PMCID: PMC11308622 DOI: 10.1186/s12885-024-12677-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Accepted: 07/23/2024] [Indexed: 08/10/2024] Open
Abstract
Acute lymphoblastic leukemia (ALL), a leading cause of childhood cancer, targets immune system B and T cells. While understanding its causes is crucial, predicting susceptibility holds immense power for early diagnosis and intervention. This study explored the potential of interleukin 10 (IL-10), a key immune regulator, as a predictive tool in Egyptian children. Investigating 100 ALL patients and 100 healthy controls, we analyzed the IL10 gene polymorphism (-1082 A/G) and serum levels. Strikingly, both the G allele and higher serum IL-10 levels were significantly associated with increased ALL risk (p < 0.05, OR > 1). Moreover, IL-10 emerged as a remarkably accurate predictor, boasting an AUC of 0.995, with a sensitivity of 97% and specificity of 96%. These findings unveil the potential of IL-10 as a powerful predictive tool for pediatric ALL in the studied Egyptian population. Identifying individuals with the GG/AG haplotype and elevated IL-10 levels could enable early intervention and potentially improve outcomes. While further validation in larger and more diverse populations is needed, this study paves the way for personalized risk assessment and potentially revolutionizes how we combat this childhood killer.
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Affiliation(s)
- Roqaia E Radwan
- Physiology Section, Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt.
| | - Ahmad Darwish
- Hematology, Oncology and Bone Marrow Transplantation Unit, Pediatric Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt
| | - Afaf M Elsaid
- Genetics Unit, Children Hospital, Mansoura University, Mansoura, Egypt
| | - Wafaa M El-Kholy
- Physiology Section, Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt
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Wang Y, Peng Q, Liu Y, Wu N, He Y, Cui X, Dan T. Genomic and transcriptomic analysis of genes involved in exopolysaccharide biosynthesis by Streptococcus thermophilus IMAU20561 grown on different sources of nitrogen. Front Microbiol 2024; 14:1328824. [PMID: 38348305 PMCID: PMC10859522 DOI: 10.3389/fmicb.2023.1328824] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2023] [Accepted: 12/31/2023] [Indexed: 02/15/2024] Open
Abstract
Exopolysaccharides (EPSs), which are produced by lactic acid bacteria, have been found to improve the texture and functionality of fermented dairy products. In a previous study, four nitrogen sources were identified as affecting the yield, molecular weight and structure of EPSs produced by Streptococcus thermophilus IMAU20561 in M17 medium. In this genomic and transcriptomics study, a novel eps gene cluster responsible for assembly of repeating units of EPS is reported. This eps cluster (22.3 kb), consisting of 24 open reading frames, is located in the chromosomal DNA. To explore the biosynthetic mechanisms in EPS, we completed RNA-seq analysis of S. thermophilus IMAU20561 grown in four different nitrogen sources for 5 h (log phase) or 10 h (stationary phase). GO functional annotation showed that there was a significant enrichment of differentially expressed genes (DEGs) involved in: amino acid biosynthesis and metabolism; ribonucleotide biosynthesis and metabolism; IMP biosynthesis and metabolism; and phosphorus metabolism. KEGG functional annotation also indicated enrichment of DEGs involved in amino acid biosynthesis, glycolysis, phosphotransferase system, fructose, and mannose metabolism. Our findings provide a better understanding the genetic traits of S. thermophilus, the biosynthetic pathways needed for the production of EPS, and a theoretical basis for screening dairy starter cultures.
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Affiliation(s)
- Yuenan Wang
- Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, China
- Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, China
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, China
| | - Qingting Peng
- Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, China
- Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, China
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, China
| | - Yang Liu
- Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, China
- Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, China
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, China
| | - Na Wu
- Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, China
- Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, China
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, China
| | - Yanyan He
- Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, China
- Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, China
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, China
| | - Xinrui Cui
- Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, China
- Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, China
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, China
| | - Tong Dan
- Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, China
- Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, China
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, China
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Shahzad A, Siddique A, Ferdous S, Amin MA, Qin M, Aslam U, Naeem M, Bashir T, Shakoor A. Heavy metals mitigation and growth promoting effect of endophytic Agrococcus terreus (MW 979614) in maize plants under zinc and nickel contaminated soil. Front Microbiol 2023; 14:1255921. [PMID: 38029198 PMCID: PMC10668838 DOI: 10.3389/fmicb.2023.1255921] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Accepted: 10/23/2023] [Indexed: 12/01/2023] Open
Abstract
Introduction Heavy metals such as iron, copper, manganese, cobalt, silver, zinc, nickel, and arsenic have accumulated in soils for a long time due to the dumping of industrial waste and sewage. Various techniques have been adapted to overcome metal toxicity in agricultural land but utilizing a biological application using potential microorganisms in heavy metals contaminated soil may be a successful approach to decontaminate heavy metals soil. Therefore, the current study aimed to isolate endophytic bacteria from a medicinal plant (Viburnum grandiflorum) and to investigate the growth-promoting and heavy metal detoxification potential of the isolated endophytic bacteria Agrococus tereus (GenBank accession number MW 979614) under nickel and zinc contamination. Methods Zinc sulfate and nickel sulfate solutions were prepared at the rate of 100 mg/kg and 50 mg/kg in sterilized distilled water. The experiment was conducted using a completely random design (CRD) with three replicates for each treatment. Results and Discussion Inoculation of seeds with A. tereus significantly increased the plant growth, nutrient uptake, and defense system. Treatment T4 (inoculated seeds), T5 (inoculated seeds + Zn100 mg/kg), and T6 (inoculated seeds + Ni 100 mg/kg) were effective, but T5 (inoculated seeds + Zn100 mg/kg) was the most pronounced and increased shoot length, root length, leaf width, plant height, fresh weight, moisture content, and proline by 49%, 38%, 89%, 31%, 113%, and 146%, respectively. Moreover the antioxidant enzymes peroxidase and super oxidase dismutase were accelerated by 211 and 68% in contaminated soil when plants were inoculated by A. tereus respectively. Similarly the inoculation of A. tereus also enhanced maize plants' absorption of Cu, Mn, Ni, Na, Cr, Fe, Ca, Mg, and K significantly. Results of the findings concluded that 100 mg/kg of Zn and Ni were toxic to maize growth, but seed inoculation with A. tereus helped the plants significantly in reducing zinc and nickel stress. The A. tereus strain may be employed as a potential strain for the detoxification of heavy metals.
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Affiliation(s)
- Asim Shahzad
- The College of Geography and Environment, Henan University, Kaifeng, China
- Department of Botany, Mohi-Ud-Din Islamic University, AJ&K, Pakistan
| | - Anam Siddique
- Department of Botany, Mohi-Ud-Din Islamic University, AJ&K, Pakistan
| | - Shazia Ferdous
- Department of Botany, Mohi-Ud-Din Islamic University, AJ&K, Pakistan
| | | | - Mingzhou Qin
- The College of Geography and Environment, Henan University, Kaifeng, China
| | - Uzma Aslam
- Department of Botany, Mohi-Ud-Din Islamic University, AJ&K, Pakistan
| | - Muhammad Naeem
- Department of Plant Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
| | - Tasmia Bashir
- Department of Botany, Rawalpindi Women University Rawalpindi, Rawalpindi, Pakistan
| | - Abdul Shakoor
- The College of Geography and Environment, Henan University, Kaifeng, China
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Codreanu SI, Ciurea CN. Candida spp. DNA Extraction in the Age of Molecular Diagnosis. Microorganisms 2023; 11:microorganisms11040818. [PMID: 37110241 PMCID: PMC10143247 DOI: 10.3390/microorganisms11040818] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2023] [Revised: 03/16/2023] [Accepted: 03/21/2023] [Indexed: 04/29/2023] Open
Abstract
The standard procedure for the detection of candidemia is blood culture, a method that might require 3-5 days for a positive result. Compared with culturing, molecular diagnosis techniques can provide faster diagnosis. The current paper aimed to present the main strengths and constraints of current molecular techniques for Candida spp. DNA extraction, analyzing their efficiency from a time, price, and ease of usage point of view. A comprehensive search was conducted using the PubMed NIH database for peer-reviewed full-text articles published before October 2022. The studies provided adequate data on the diagnosis of the infection with the Candida spp. DNA extraction is a relevant step in yielding pure qualitative DNA to be amplified in molecular diagnostic techniques. The most used fungal DNA extraction strategies are: mechanical (bead beating, ultrasonication, steel-bullet beating), enzymatic (proteinase K, lysozyme, lyticase), and chemical extraction (formic acid, liquid nitrogen, ammonium chloride). More clinical studies are needed to formulate adequate guidelines for fungal DNA extraction as the current paper highlighted discrepancies in the reported outcome.
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Affiliation(s)
- Smaranda Ioana Codreanu
- Faculty of Medicine, "George Emil Palade" University of Medicine, Pharmacy, Sciences and Technology of Târgu Mures, 38 Gheorghe Marinescu Street, 540139 Târgu Mures, Romania
| | - Cristina Nicoleta Ciurea
- Department of Microbiology, Faculty of Medicine, "George Emil Palade" University of Medicine, Pharmacy, Sciences and Technology of Târgu Mures, 38 Gheorghe Marinescu Street, 540139 Târgu Mures, Romania
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Nguyen HA, Lee NY. Copper: DNA extraction and solid phase detection agent for all-in-one molecular diagnostic device coupled with isothermal amplification. Biosens Bioelectron 2023; 229:115222. [PMID: 36989581 DOI: 10.1016/j.bios.2023.115222] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2022] [Revised: 01/28/2023] [Accepted: 03/09/2023] [Indexed: 03/14/2023]
Abstract
In this study, an all-in-one poly(methyl methacrylate) (PMMA) device integrating two novel techniques - DNA extraction employing a CuSO4/H2O2 system and DNA detection utilizing solid phase copper tape - coupled with loop-mediated isothermal amplification (LAMP) is developed for on-site pathogen detection. The CuSO4/H2O2 system, also known as Fenton-like reaction, is used to produce hydroxyl radicals, which can disrupt bacterial membranes via lipid peroxidation and release DNA at room temperature. The released DNA is subsequently amplified by LAMP reaction. The acidic environment resulting from the production of hydrogen ions in the presence of target DNA in the LAMP reaction can stimulate the color change on copper tape due to the corrosion, while the innate alkaline environment in a negative sample not containing target DNA cannot stimulate the corrosion. The fabricated PMMA device integrates all the functionalities necessary for molecular diagnostics such as DNA extraction, amplification, and detection, and a carbon paste-based heater is fabricated for LAMP reaction. Using the PMMA device, Enterococcus faecium was detected as low as 4.67 × 102 CFU/mL within 90 min. E. faecium spiked in milk was successfully detected using the all-in-one PMMA device. The equipment-free techniques for decentralized diagnostics and naked-eye readout of results coupled with the portable heater serves as a promising solution for point-of-care testing particularly in a resource-limited environment.
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Affiliation(s)
- Hanh An Nguyen
- Department of BioNano Technology, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam-si, Gyeonggi-do, 13120, South Korea
| | - Nae Yoon Lee
- Department of BioNano Technology, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam-si, Gyeonggi-do, 13120, South Korea.
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7
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Chopra S, Kumar D. Characteristics and growth kinetics of biomass of Citrobacter freundii strains PYI-2 and Citrobacter portucalensis strain YPI-2 during the biodegradation of Ibuprofen. Int Microbiol 2022; 25:615-628. [PMID: 35553276 DOI: 10.1007/s10123-022-00248-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Revised: 04/14/2022] [Accepted: 04/28/2022] [Indexed: 11/25/2022]
Abstract
Ibuprofen (IBU) is the third most commonly used analgesic drug in the world. It enters the water system as a result of human excretion-based wastewater discharges. Hence, it attracts the attention of environmentalists for its ecological fate and degradation behavior. In this study, the two IBU degrading bacterial strains, Citrobacter freundii strain PYI-2 (MT039504) and Citrobacter portucalensis strain YPI-2 (MN744335), were isolated from industrial wastewater samples using an enrichment culture method, identified, and characterized. Physiological and batch culture degradation studies have indicated that these strains involved in IBU degradation and the intermediates produced during the process were analyzed. These strains degrade IBU in the batch culture. The optimum pH was reported for degradation of the PYI2 strain (6.9) and YPI2 strain (5.8), and the optimum temperatures were 42°C and 32°C, respectively. Biomass kinetic analysis of these strains was performed based on physical parameters (temperature, pH, and rpm) and confirmed by the experimental study. As indicated in the GC-MS chromatogram peaks, viz., hydroxyibuprofen, 2-(4-hydroxyphenylpropionic acid), 1,4-hydroquinone, and 2-hydroxy-1,4-quinol various intermediates compounds of degradation pathway were observed. Finally, through the GC-MS data, the metabolic pathway for degradation was predicted. In the study, it was confirmed that Citrobacter freundii strain PYI-2 and Citrobacter portucalensis strain YPI-2 exhibit metabolic potential for the biodegradation of IBU and can be further deployed in bioremediation.
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Affiliation(s)
- Sunil Chopra
- Department of Biotechnology, Deenbandhu Chhotu Ram University of Science and Technology, Murthal, Sonepat, Haryana, 131039, India
| | - Dharmender Kumar
- Department of Biotechnology, Deenbandhu Chhotu Ram University of Science and Technology, Murthal, Sonepat, Haryana, 131039, India.
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Högberg N, Baltrušis P, Enweji N, Höglund J. Assessment of three DNA extraction kits for the absolute quantification of strongyle nematode eggs in faecal samples. Acta Vet Scand 2022; 64:5. [PMID: 35139862 PMCID: PMC8826664 DOI: 10.1186/s13028-022-00624-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Accepted: 01/26/2022] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes of small ruminants. The current diagnostic approach for the detection of this species relies on coproscopic methods, which both have low sensitivity and are time consuming. Methods employing detection through DNA amplification, such as droplet digital polymerase chain reaction (ddPCR), offer an advantageous approach to the diagnosis of H. contortus. However, DNA extraction protocols need to be constantly updated for the optimal retrieval of diagnostically usable template. Here, we describe the evaluation of three genomic DNA extraction kits for the detection and quantification of H. contortus ITS2 amplicon DNA from faecal samples, using droplet digital PCR. RESULTS DNA samples, extracted from faecal material with the Nucleospin DNA Stool kit, produced the highest amounts of ITS2 amplicon copies and had the lowest coefficient of variation across different dilutions and sample types (fresh or frozen) out of the tested kits (Nucleospin DNA Stool, E.Z.N.A.® Stool DNA Kit and QIAamp Fast DNA Stool Mini Kit). Furthermore, the protocol of this kit has the fewest number of steps and the price of DNA extraction per sample is reasonable (2.77 €). CONCLUSIONS The Nucleospin DNA Stool kit is an attractive option for the detection and quantification of H. contortus DNA in faecal samples of small ruminants in a diagnostic setting.
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Affiliation(s)
- Niclas Högberg
- Parasitology Unit, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7036, 750 07 Uppsala, Sweden
| | - Paulius Baltrušis
- Parasitology Unit, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7036, 750 07 Uppsala, Sweden
| | - Nizar Enweji
- Parasitology Unit, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7036, 750 07 Uppsala, Sweden
| | - Johan Höglund
- Parasitology Unit, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7036, 750 07 Uppsala, Sweden
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Busi SB, Pramateftaki P, Brandani J, Fodelianakis S, Peter H, Halder R, Wilmes P, Battin TJ. Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams. PeerJ 2020; 8:e9973. [PMID: 33194372 PMCID: PMC7597623 DOI: 10.7717/peerj.9973] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2020] [Accepted: 08/26/2020] [Indexed: 11/20/2022] Open
Abstract
Glacier-fed streams (GFS) are harsh ecosystems dominated by microbial life organized in benthic biofilms, yet the biodiversity and ecosystem functions provided by these communities remain under-appreciated. To better understand the microbial processes and communities contributing to GFS ecosystems, it is necessary to leverage high throughput sequencing. Low biomass and high inorganic particle load in GFS sediment samples may affect nucleic acid extraction efficiency using extraction methods tailored to other extreme environments such as deep-sea sediments. Here, we benchmarked the utility and efficacy of four extraction protocols, including an up-scaled phenol-chloroform protocol. We found that established protocols for comparable sample types consistently failed to yield sufficient high-quality DNA, delineating the extreme character of GFS. The methods differed in the success of downstream applications such as library preparation and sequencing. An adapted phenol-chloroform-based extraction method resulted in higher yields and better recovered the expected taxonomic profile and abundance of reconstructed genomes when compared to commercially-available methods. Affordable and straight-forward, this method consistently recapitulated the abundance and genomes of a mock community, including eukaryotes. Moreover, by increasing the amount of input sediment, the protocol is readily adjustable to the microbial load of the processed samples without compromising protocol efficiency. Our study provides a first systematic and extensive analysis of the different options for extraction of nucleic acids from glacier-fed streams for high-throughput sequencing applications, which may be applied to other extreme environments.
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Affiliation(s)
- Susheel Bhanu Busi
- Systems Ecology Research Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg
| | - Paraskevi Pramateftaki
- Stream Biofilm and Ecosystems Research group, École Polytechnique Federale de Lausanne, Lausanne, Switzerland
| | - Jade Brandani
- Stream Biofilm and Ecosystems Research group, École Polytechnique Federale de Lausanne, Lausanne, Switzerland
| | - Stilianos Fodelianakis
- Stream Biofilm and Ecosystems Research group, École Polytechnique Federale de Lausanne, Lausanne, Switzerland
| | - Hannes Peter
- Stream Biofilm and Ecosystems Research group, École Polytechnique Federale de Lausanne, Lausanne, Switzerland
| | - Rashi Halder
- Systems Ecology Research Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg
| | - Paul Wilmes
- Systems Ecology Research Group, Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg
| | - Tom J Battin
- Stream Biofilm and Ecosystems Research group, École Polytechnique Federale de Lausanne, Lausanne, Switzerland
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10
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A robust, hand-powered, instrument-free sample preparation system for point-of-care pathogen detection. Sci Rep 2019; 9:16374. [PMID: 31705044 PMCID: PMC6841715 DOI: 10.1038/s41598-019-52922-y] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2019] [Accepted: 10/25/2019] [Indexed: 11/08/2022] Open
Abstract
Here, we describe a simple, universal protocol for use in nucleic acid testing-based pathogen diagnostics, which requires only hand-powered sample preparation, including the processes of pathogen enrichment and nucleic acid isolation. The protocol uses low-cost amine-functionalized diatomaceous earth with a 1-μm Teflon filter as a reaction matrix in both stages of the process, using homobifunctional imidoesters. Using a simple syringe as a pump, the capture efficiency for a large sample volume (<50 mL) was enhanced by up to 98.3%, and the detection limit was 1 CFU/mL, 100-fold better than that of common commercial nucleic acid isolation kit. This protocol can also be combined with commercialized 96-well filter plates for robust sample preparation. Our proposed system is robust, simple, low-cost, universal, and rapid (taking <20 min), and it works regardless of the ambient environment and sample pretreatment, requiring no electricity or instruments. Its benefits include the simplicity of producing its components and its ease of operation, and it can be readily integrated with other assays for point-of-care diagnostics.
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11
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Dunbar J, Gallegos-Graves LV, Gans J, Morse SA, Pillai S, Anderson K, Hodge DR. Evaluation of DNA extraction methods to detect bacterial targets in aerosol samples. J Microbiol Methods 2018; 153:48-53. [PMID: 30201412 DOI: 10.1016/j.mimet.2018.09.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Revised: 09/06/2018] [Accepted: 09/06/2018] [Indexed: 11/29/2022]
Abstract
DNA-based monitoring of pathogens in aerosol samples requires extraction methods that provide high recovery of DNA. To identify a suitable method, we evaluated six DNA extraction methods for recovery of target-specific DNA from samples with four bacterial agents at low abundance (<10,000 genome copies per detection assay). These methods differed in rigor of cell disruption, approach for DNA capture, and extent of DNA purification. The six methods varied 1000-fold in the recovery of DNA from spores or cells of surrogates of Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei, and Francisella tularensis, each at about 105 CFU per sample. A custom method using paramagnetic Dynabeads for DNA capture greatly outperformed the other five methods. The cDynabead method provided about 80% recovery of target-specific DNA. The cDynabead method and a filtration method were further evaluated for DNA recovery from bacterial agents spiked on filters (c.a. 105 CFU of each agent per filter quadrant) that were subsequently used to collect background outdoor air particulates for 24-h. The filtration method generally failed to recover detectable quantities of target DNA from the spiked filters, suggesting at least a 100-fold loss of target DNA during extraction, whereas the custom cDynabead method consistently yielded DNA sufficient for target detection.
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Affiliation(s)
- John Dunbar
- Los Alamos National Laboratory, Los Alamos, NM, United States.
| | | | - Jason Gans
- Los Alamos National Laboratory, Los Alamos, NM, United States
| | | | - Segaran Pillai
- Food and Drug Administration, Washington, DC, United States
| | - Kevin Anderson
- Department of Homeland Security, Washington, DC, United States
| | - David R Hodge
- Department of Homeland Security, Washington, DC, United States
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12
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Abstract
Nucleic acid extraction is the first step of any amplification experiment no matter what kind of amplification is used to detect a specific pathogen. Efficient nucleic acid extraction is essential to obtain good results using any molecular test. The optimal extraction method should fulfill the following conditions: speed, short working time, cost-effectiveness, high sensitivity and specificity, good reproducibility, and safety. The methods can be divided into solution or column based according to differences of their principles. The automated extraction instruments have many advantages, and these have proven to be very useful. Moreover, in recent years, fully automated instruments combining NA extraction and amplification have been commercially available. However, the method itself does not provide assurance, and the DNA recovery can be different among various kits or instruments that use the similar principles. Therefore, it is important to carefully evaluate the performance of any extraction method used in the clinical microbiology laboratory even though manufacturers may have reported good validation results with specific organisms.
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Affiliation(s)
- Yi-Wei Tang
- Departments of Laboratory Medicine and Internal Medicine, Memorial Sloan Kettering Cancer Center, New York, NY USA
| | - Charles W. Stratton
- Department of Pathology, Microbiology and Immunology and Medicine, Vanderbilt University Medical Center, Nashville, TN USA
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13
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Quainoo S, Coolen JPM, van Hijum SAFT, Huynen MA, Melchers WJG, van Schaik W, Wertheim HFL. Whole-Genome Sequencing of Bacterial Pathogens: the Future of Nosocomial Outbreak Analysis. Clin Microbiol Rev 2017; 30:1015-1063. [PMID: 28855266 PMCID: PMC5608882 DOI: 10.1128/cmr.00016-17] [Citation(s) in RCA: 269] [Impact Index Per Article: 33.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Outbreaks of multidrug-resistant bacteria present a frequent threat to vulnerable patient populations in hospitals around the world. Intensive care unit (ICU) patients are particularly susceptible to nosocomial infections due to indwelling devices such as intravascular catheters, drains, and intratracheal tubes for mechanical ventilation. The increased vulnerability of infected ICU patients demonstrates the importance of effective outbreak management protocols to be in place. Understanding the transmission of pathogens via genotyping methods is an important tool for outbreak management. Recently, whole-genome sequencing (WGS) of pathogens has become more accessible and affordable as a tool for genotyping. Analysis of the entire pathogen genome via WGS could provide unprecedented resolution in discriminating even highly related lineages of bacteria and revolutionize outbreak analysis in hospitals. Nevertheless, clinicians have long been hesitant to implement WGS in outbreak analyses due to the expensive and cumbersome nature of early sequencing platforms. Recent improvements in sequencing technologies and analysis tools have rapidly increased the output and analysis speed as well as reduced the overall costs of WGS. In this review, we assess the feasibility of WGS technologies and bioinformatics analysis tools for nosocomial outbreak analyses and provide a comparison to conventional outbreak analysis workflows. Moreover, we review advantages and limitations of sequencing technologies and analysis tools and present a real-world example of the implementation of WGS for antimicrobial resistance analysis. We aimed to provide health care professionals with a guide to WGS outbreak analysis that highlights its benefits for hospitals and assists in the transition from conventional to WGS-based outbreak analysis.
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Affiliation(s)
- Scott Quainoo
- Department of Microbiology, Radboud University, Nijmegen, The Netherlands
| | - Jordy P M Coolen
- Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands
| | - Sacha A F T van Hijum
- Centre for Molecular and Biomolecular Informatics, Radboud University Medical Centre, Nijmegen, The Netherlands
- NIZO, Ede, The Netherlands
| | - Martijn A Huynen
- Centre for Molecular and Biomolecular Informatics, Radboud University Medical Centre, Nijmegen, The Netherlands
| | - Willem J G Melchers
- Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands
| | - Willem van Schaik
- Institute of Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom
| | - Heiman F L Wertheim
- Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands
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14
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Dalla-Costa LM, Morello LG, Conte D, Pereira LA, Palmeiro JK, Ambrosio A, Cardozo D, Krieger MA, Raboni SM. Comparison of DNA extraction methods used to detect bacterial and yeast DNA from spiked whole blood by real-time PCR. J Microbiol Methods 2017; 140:61-66. [PMID: 28669799 DOI: 10.1016/j.mimet.2017.06.020] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2017] [Revised: 06/27/2017] [Accepted: 06/29/2017] [Indexed: 12/16/2022]
Abstract
Sepsis is the leading cause of death in intensive care units (ICUs) worldwide and its diagnosis remains a challenge. Blood culturing is the gold standard technique for blood stream infection (BSI) identification. Molecular tests to detect pathogens in whole blood enable early use of antimicrobials and affect clinical outcomes. Here, using real-time PCR, we evaluated DNA extraction using seven manual and three automated commercially available systems with whole blood samples artificially contaminated with Escherichia coli, Staphylococcus aureus, and Candida albicans, microorganisms commonly associated with BSI. Overall, the commercial kits evaluated presented several technical limitations including long turnaround time and low DNA yield and purity. The performance of the kits was comparable for detection of high microorganism loads (106CFU/mL). However, the detection of lower concentrations was variable, despite the addition of pre-processing treatment to kits without such steps. Of the evaluated kits, the UMD-Universal CE IVD kit generated a higher quantity of DNA with greater nucleic acid purity and afforded the detection of the lowest microbial load in the samples. The inclusion of pre-processing steps with the kit seems to be critical for the detection of microorganism DNA directly from whole blood. In conclusion, future application of molecular techniques will require overcoming major challenges such as the detection of low levels of microorganism nucleic acids amidst the large quantity of human DNA present in samples or differences in the cellular structures of etiological agents that can also prevent high-quality DNA yields.
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Affiliation(s)
- Libera M Dalla-Costa
- Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil; Laboratory of Bacteriology, Universidade Federal do Paraná, Rua Padre Camargo, 280, - 80060-240, Curitiba, Brazil; Faculdades e Instituto de Pesquisa Pelé Pequeno Príncipe, Av. Silva Jardim, 1632, - 80250-200, Curitiba, Brazil
| | - Luis G Morello
- Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil; Laboratory of Functional Genomics, Instituto Carlos Chagas, Fundação Oswaldo Cruz, Rua Professor Algacyr Munhoz Mader, 3775, - 81310-020, Curitiba, Brazil
| | - Danieli Conte
- Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil
| | - Luciane A Pereira
- Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil
| | - Jussara K Palmeiro
- Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil; Laboratory of Bacteriology, Universidade Federal do Paraná, Rua Padre Camargo, 280, - 80060-240, Curitiba, Brazil
| | - Altair Ambrosio
- Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil; Laboratory of Bacteriology, Universidade Federal do Paraná, Rua Padre Camargo, 280, - 80060-240, Curitiba, Brazil
| | - Dayane Cardozo
- Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil
| | - Marco A Krieger
- Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil; Laboratory of Functional Genomics, Instituto Carlos Chagas, Fundação Oswaldo Cruz, Rua Professor Algacyr Munhoz Mader, 3775, - 81310-020, Curitiba, Brazil.
| | - Sonia M Raboni
- Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader, 3775, - 81925-610, Curitiba, Brazil; Infectious Disease Division, Universidade Federal do Paraná, Rua Gen. Carneiro, 181, - 80060-900, Curitiba, Brazil.
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15
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Evaluation of chest radiography, lytA real-time PCR, and other routine tests for diagnosis of community-acquired pneumonia and estimation of possible attributable fraction of pneumococcus in northern Togo. Epidemiol Infect 2016; 145:583-594. [PMID: 27852346 PMCID: PMC5244441 DOI: 10.1017/s0950268816002211] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Streptococcus pneumoniae (Spn) is a leading cause of community-acquired pneumonia (CAP), yet existing diagnostic tools remain inadequate. We aimed to evaluate laboratory and radiological methods for detecting pneumococcal aetiology in CAP patients and to estimate Spn prevalence in this group. All-aged patients hospitalized with clinically defined CAP in northern Togo were enrolled during 2010–2013. Latent class analysis pooled results of semi-automated blood culture (SABC), whole blood lytA real-time polymerase chain reaction (rt-PCR), serum C-reactive protein (CRP), and chest radiography (CXR) and categorized patients as likely pneumococcal or non-pneumococcal CAP. We enrolled 1684 patients; 1501 had results for all tests. CXR, SABC, lytA rt-PCR and CRP >71·2 mg/l had sensitivities of 94% [95% confidence interval (CI) 87–100], 13% (95% CI 10–16), 17% (95% CI 14–21) and 78% (95% CI 75–80), and specificities of 88% (95% CI 84–93), 100% (95% CI 99–100), 97% (95% CI 96–99) and 77% (95% CI 75–79), respectively. Pneumococcal attributable proportion was 34% (95% CI 32–37), increasing with age and in men. We estimated that Spn caused one third of CAP. Whole blood lytA rt-PCR was more sensitive than SABC; both had low sensitivity and high specificity. Conversely CXR was highly sensitive and reasonably specific; it could be a useful tool for epidemiological studies aiming to define Spn pneumonia incidence across all ages.
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16
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Mölsä M, Kalin-Mänttäri L, Tonteri E, Hemmilä H, Nikkari S. Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples. J Microbiol Methods 2016; 128:69-73. [PMID: 27435532 DOI: 10.1016/j.mimet.2016.07.013] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Revised: 07/15/2016] [Accepted: 07/15/2016] [Indexed: 11/28/2022]
Abstract
Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity.
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Affiliation(s)
- Markos Mölsä
- Centres for Military Medicine and for Biological Threat Preparedness, Helsinki, Finland.
| | | | - Elina Tonteri
- Centres for Military Medicine and for Biological Threat Preparedness, Helsinki, Finland
| | - Heidi Hemmilä
- Centres for Military Medicine and for Biological Threat Preparedness, Helsinki, Finland
| | - Simo Nikkari
- Centres for Military Medicine and for Biological Threat Preparedness, Helsinki, Finland
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17
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Nieman AE, Savelkoul PHM, Beishuizen A, Henrich B, Lamik B, MacKenzie CR, Kindgen-Milles D, Helmers A, Diaz C, Sakka SG, Schade RP. A prospective multicenter evaluation of direct molecular detection of blood stream infection from a clinical perspective. BMC Infect Dis 2016; 16:314. [PMID: 27364885 PMCID: PMC4928256 DOI: 10.1186/s12879-016-1646-4] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2015] [Accepted: 06/10/2016] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Rapid diagnosis and appropriate antimicrobial therapy are of major importance to decrease morbidity and mortality in patients with blood stream infections (BSI). Blood culture, the current gold standard for detecting bacteria in blood, requires at least 24-48 hours and has limited sensitivity if obtained during antibiotic treatment of the patient. The aim of this prospective multicenter study was to clinically evaluate the application of a commercial universal 16S/18S rDNA PCR, SepsiTest™ (PCR-ST), directly on whole blood. METHODS In total 236 samples from 166 patients with suspected sepsis were included in the study. PCR-ST results were compared to blood culture, the current gold standard for detecting BSI. Because blood cultures can give false-negative results, we performed an additional analysis to interpret the likelihood of bloodstream infection by using an evaluation based on clinical diagnosis, other diagnostic tests and laboratory parameters. RESULTS Clinical interpretation of results defined the detected organism to be contaminants in 22 of 43 positive blood cultures (51.2 %) and 21 of 47 positive PCR-ST results (44.7 %). Excluding these contaminants resulted in an overall sensitivity and specificity of the PCR-ST of 66.7 and 94.4 % respectively. Of the 36 clinically relevant samples, 11 BSI were detected with both techniques, 15 BSI were detected with PCR-ST only and 10 with blood culture only. Therefore, in this study, SepsiTest™ detected an additional 71 % BSI compared to blood culture alone. CONCLUSIONS More clinically relevant BSI were diagnosed by molecular detection, which might influence patient treatment. An improved SepsiTest™ assay suited for routine use can have additional value to blood culture in diagnosing bacteremia in septic patients.
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Affiliation(s)
- A. E. Nieman
- />Department of Medical Microbiology and Infection control, VU University Medical Center, Amsterdam, The Netherlands
- />Laboratory for Medical Microbiology, Immunology and Infection Control, Elisabeth-TweeSteden Hospital, Tilburg, The Netherlands
| | - P. H. M. Savelkoul
- />Department of Medical Microbiology and Infection control, VU University Medical Center, Amsterdam, The Netherlands
- />Department of Medical Microbiology, Maastricht University Medical Center, Maastricht, The Netherlands
| | - A. Beishuizen
- />Department of Intensive Care Medicine, VU University Medical Center, Amsterdam, The Netherlands
- />Department of Intensive Care, Medisch Spectrum Twente, Enschede, The Netherlands
| | - B. Henrich
- />Institute of Medical Microbiology and Hospital Hygiene, University Clinic of Heinrich-Heine University Düsseldorf, Düsseldorf, Germany
| | - B. Lamik
- />Institute of Medical Microbiology and Hospital Hygiene, University Clinic of Heinrich-Heine University Düsseldorf, Düsseldorf, Germany
| | - C. R. MacKenzie
- />Institute of Medical Microbiology and Hospital Hygiene, University Clinic of Heinrich-Heine University Düsseldorf, Düsseldorf, Germany
| | - D. Kindgen-Milles
- />Department of Anesthesiology, University Clinic of Heinrich-Heine University Düsseldorf, Düsseldorf, Germany
| | - A. Helmers
- />Department of Microbiology, Merheim Zentrallabor, MVZ synlab Leverkusen GmbH, Leverkusen, Germany
| | - C. Diaz
- />Department of Microbiology, Merheim Zentrallabor, MVZ synlab Leverkusen GmbH, Leverkusen, Germany
| | - S. G. Sakka
- />Department of Anesthesiology and Intensive Care Medicine, Medical Center Cologne-Merheim, University Witten/Herdecke, Cologne, Germany
| | - R. P. Schade
- />Department of Medical Microbiology and Infection control, VU University Medical Center, Amsterdam, The Netherlands
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Gaignaux A, Ashton G, Coppola D, De Souza Y, De Wilde A, Eliason J, Grizzle W, Guadagni F, Gunter E, Koppandi I, Shea K, Shi T, Stein JA, Sobel ME, Tybring G, Van den Eynden G, Betsou F. A Biospecimen Proficiency Testing Program for Biobank Accreditation: Four Years of Experience. Biopreserv Biobank 2016; 14:429-439. [PMID: 27195612 DOI: 10.1089/bio.2015.0108] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Biobanks produce and distribute biospecimens, ensuring their fitness for purpose and accurately qualifying them before distribution. In their efforts toward professionalization, biobanks can nowadays seek certification or accreditation. One of the requirements of these standards is regular participation in Proficiency Testing (PT) programs. An international PT program has been developed and provided to biobanks and other laboratories that perform specific tests to qualify different types of biospecimens. This PT program includes biospecimen testing schemes, as well as biospecimen processing interlaboratory exercises. This PT program supports the development of biobank quality assurance by providing the possibility to assess biobank laboratory performance and useful insights into biobank laboratory method performance characteristics and thus fulfill the demands from accreditation authorities.
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Affiliation(s)
| | - Garry Ashton
- 2 Cancer Research UK Manchester Institute , Manchester, United Kingdom
| | | | - Yvonne De Souza
- 4 AIDS Specimen Bank, University of California , San Francisco, San Francisco, California
| | | | - James Eliason
- 6 Great Lakes Stem Cell Innovation Center , Detroit, Michigan
| | - William Grizzle
- 7 Tissue Collection and Banking Facility, University of Alabama , Birmingham, Birmingham, Alabama
| | - Fiorella Guadagni
- 8 BioBIM (Multidisciplinary Interinstitutional Biobank) IRCCS San Raffaele , Rome, Italy
| | | | - Iren Koppandi
- 10 Cellular Technology Limited , Shaker Heights, Ohio
| | | | - Tim Shi
- 12 GlobalMD Network Corporation , Catonsville, Maryland
| | - Julie A Stein
- 13 PPD Vaccines and Biologics Lab , Wayne, Pennsylvania
| | - Mark E Sobel
- 14 American Society for Investigative Pathology , Bethesda, Maryland
| | | | - Gert Van den Eynden
- 16 Molecular Immunology Unit, Institut Jules Bordet , Brussels, Belgium .,17 Pathobiology Group , EORTC, Brussels, Belgium
| | - Fay Betsou
- 1 Integrated Biobank of Luxembourg , Luxembourg, Luxembourg
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19
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Xia LP, Bian LY, Xu M, Liu Y, Tang AL, Ye WQ. 16S rRNA gene sequencing is a non-culture method of defining the specific bacterial etiology of ventilator-associated pneumonia. Int J Clin Exp Med 2015; 8:18560-18570. [PMID: 26770469 PMCID: PMC4694369] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2015] [Accepted: 10/13/2015] [Indexed: 06/05/2023]
Abstract
Ventilator-associated pneumonia (VAP) is an acquired respiratory tract infection following tracheal intubation. The most common hospital-acquired infection among patients with acute respiratory failure, VAP is associated with a mortality rate of 20-30%. The standard bacterial culture method for identifying the etiology of VAP is not specific, timely, or accurate in identifying the bacterial pathogens. This study used 16S rRNA gene metagenomic sequencing to identify and quantify the pathogenic bacteria in lower respiratory tract and oropharyngeal samples of 55 VAP patients. Sequencing of the 16S rRNA gene has served as a valuable tool in bacterial identification, particularly when other biochemical, molecular, or phenotypic identification techniques fail. In this study, 16S rRNA gene sequencing was performed in parallel with the standard bacterial culture method to identify and quantify bacteria present in the collected patient samples. Sequence analysis showed the colonization of multidrug-resistant strains in VAP secretions. Further, this method identified Prevotella, Proteus, Aquabacter, and Sphingomonas bacterial genera that were not detected by the standard bacterial culture method. Seven categories of bacteria, Streptococcus, Neisseria, Corynebacterium, Acinetobacter, Staphylococcus, Pseudomonas and Klebsiella, were detectable by both 16S rRNA gene sequencing and standard bacterial culture methods. Further, 16S rRNA gene sequencing had a significantly higher sensitivity in detecting Streptococcus and Pseudomonas when compared to standard bacterial culture. Together, these data present 16S rRNA gene sequencing as a novel VAP diagnosis tool that will further enable pathogen-specific treatment of VAP.
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Affiliation(s)
- Li-Ping Xia
- Department of Nursing, Changhai Hospital, Second Military Medical UniversityShanghai 200433, P. R. China
- Department of Nursing, Yancheng Health Vocational and Technical CollegeYancheng 224006, Jiangsu Province, P. R. China
| | - Long-Yan Bian
- Department of Nursing, Yancheng Health Vocational and Technical CollegeYancheng 224006, Jiangsu Province, P. R. China
| | - Min Xu
- Yancheng First People’s HospitalYancheng 224006, Jiangsu Province, P. R. China
| | - Ying Liu
- Department of Nursing, Changhai Hospital, Second Military Medical UniversityShanghai 200433, P. R. China
| | - Ai-Ling Tang
- Department of Nursing, Changhai Hospital, Second Military Medical UniversityShanghai 200433, P. R. China
| | - Wen-Qin Ye
- Department of Nursing, Changhai Hospital, Second Military Medical UniversityShanghai 200433, P. R. China
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20
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Psifidi A, Dovas CI, Bramis G, Lazou T, Russel CL, Arsenos G, Banos G. Comparison of eleven methods for genomic DNA extraction suitable for large-scale whole-genome genotyping and long-term DNA banking using blood samples. PLoS One 2015; 10:e0115960. [PMID: 25635817 PMCID: PMC4312062 DOI: 10.1371/journal.pone.0115960] [Citation(s) in RCA: 86] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2014] [Accepted: 11/28/2014] [Indexed: 12/21/2022] Open
Abstract
Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.
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Affiliation(s)
- Androniki Psifidi
- Animal Production Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
- The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom
- * E-mail:
| | - Chrysostomos I. Dovas
- Microbiology and Infectious Diseases Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Georgios Bramis
- Animal Production Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Thomai Lazou
- Food safety Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Claire L. Russel
- Department of Clinical Veterinary Sciences, University of Bristol, Langford House, Langford, Bristol, United Kingdom
| | - Georgios Arsenos
- Animal Production Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Georgios Banos
- Animal Production Laboratory, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
- The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom
- Scotland’s Rural College, Edinburgh, United Kingdom
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21
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Ince J, McNally A. Development of rapid, automated diagnostics for infectious disease: advances and challenges. Expert Rev Med Devices 2014; 6:641-51. [DOI: 10.1586/erd.09.46] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
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22
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Gosiewski T, Szała L, Pietrzyk A, Brzychczy-Włoch M, Heczko PB, Bulanda M. Comparison of methods for isolation of bacterial and fungal DNA from human blood. Curr Microbiol 2013; 68:149-55. [PMID: 24026449 PMCID: PMC3895209 DOI: 10.1007/s00284-013-0451-1] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2013] [Accepted: 08/09/2013] [Indexed: 11/23/2022]
Abstract
The study aimed at optimization of DNA isolation from blood of representatives of four microbial groups causing sepsis, i.e., Gram negative: Escherichia coli, Gram positive: Staphylococcus aureus, yeast: Candida albicans, and filamentous fungus: Aspergillus fumigatus. Additionally, the five commercial kits for microbial DNA isolation from the blood were tested. The developed procedure of DNA isolation consisted of three consecutive steps, i.e., mechanical disruption, chemical lysis, and thermal lysis. Afterward, DNA was isolated from the previously prepared samples (erythrocyte lysis) with the use of five commercial kits for DNA isolation. They were compared paying heed to detection limit, concentration, DNA purity, and heme concentration in samples. The isolation of DNA without preliminary erythrocyte lysis resulted in far higher heme concentration than when lysis was applied. In the variant with erythrocyte lysis, two of the commercial kits were most effective in purifying the DNA extract from heme. Designed procedure allowed obtaining microbial DNA from all four groups of pathogens under study in the amount sufficient to conduct the rtPCR reaction, which aimed at detecting them in the blood.
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Affiliation(s)
- Tomasz Gosiewski
- Chair of Microbiology, Jagiellonian University Medical College, 18 Czysta Str., 31-121, Kraków, Poland,
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23
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Karlsson OE, Hansen T, Knutsson R, Löfström C, Granberg F, Berg M. Metagenomic Detection Methods in Biopreparedness Outbreak Scenarios. Biosecur Bioterror 2013; 11 Suppl 1:S146-57. [DOI: 10.1089/bsp.2012.0077] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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Hansen WLJ, Bruggeman CA, Wolffs PFG. Pre-analytical sample treatment and DNA extraction protocols for the detection of bacterial pathogens from whole blood. Methods Mol Biol 2013; 943:81-90. [PMID: 23104282 DOI: 10.1007/978-1-60327-353-4_4] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
Molecular diagnostics is an increasing popular approach for the direct detection and identification of pathogenic bacteria in clinical samples. Conventional culture techniques are time-consuming and therefore causing a delay in the diagnosis of the patient. Alternative techniques based on nucleic acid amplification offer a shorter turn-around-time and the ability to identify fastidious and non-cultivable organisms. However, molecular detection of bacteria in blood, by for example PCR, RT-PCR, or sequencing of the 16S rDNA genes is often complicated by the presence of PCR-inhibitory compounds. Here we describe several different methods for the extraction of bacterial DNA from whole blood samples. The methods differ regarding costs, hands-on time as well as regarding sensitivity. In combination with a model PCR the detection limits that can be reached using the different methods range from 1,000 to 50 cfu/ml.
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Affiliation(s)
- Wendy L J Hansen
- Department of Medical Microbiology, Maastricht University Medical Center, Maastricht, The Netherlands
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25
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Abstract
Since thermostable Taq DNA polymerase was discovered in 1987, nucleic acid amplification techniques have made great strides and contributed greatly to progress in the life sciences. These techniques were introduced into the clinical laboratory and have produced great changes in diagnostic tools and tests. In particular, there have been many innovative molecular testing developments in the field of diagnostic microbiology.
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26
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Portilho MM, Martins PP, Lampe E, Villar LM. A comparison of molecular methods for hepatitis B virus (HBV) DNA detection from oral fluid samples. J Med Microbiol 2012; 61:844-851. [PMID: 22403138 DOI: 10.1099/jmm.0.040238-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The objective of the present study was to evaluate four commercial DNA extraction methods and three PCR protocols for hepatitis B virus (HBV) detection in artificially contaminated oral fluid samples. The extraction protocols were selected based on ease of use and cost, and were also compared with respect to sensitivity and cost. Prior PCR optimization was conducted, in which the sample volume for DNA extraction and the concentrations of DNA and Taq DNA polymerase in the PCR were adjusted. One-round PCR, used to amplify the core region of the HBV genome, achieved high levels of sensitivity in comparison with nested and semi-nested PCR experiments that were designed for the amplification of HBV surface protein genes. Of the four extraction protocols evaluated, the RTP DNA/RNA Virus Mini kit and the QIAamp DNA Mini kit gave the highest recovery rates, presenting 20 copies of HBV DNA ml(-1) as the limit of detection. These results suggest that HBV DNA can be detected from oral fluid samples but that the optimization of the PCR assays and the choice of extraction methods must be determined by laboratories before the implementation of this method in routine diagnostics.
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Affiliation(s)
| | - Patrícia Pais Martins
- Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil
| | - Elisabeth Lampe
- Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil
| | - Livia Melo Villar
- Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil
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Khokhar SK, Mitui M, Leos NK, Rogers BB, Park JY. Evaluation of Maxwell® 16 for automated DNA extraction from whole blood and formalin-fixed paraffin embedded (FFPE) tissue. Clin Chem Lab Med 2011; 50:267-72. [PMID: 22022984 DOI: 10.1515/cclm.2011.763] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2011] [Accepted: 10/05/2011] [Indexed: 11/15/2022]
Abstract
BACKGROUND The aim of the study was to assess the performance of Promega, Maxwell® 16 for the extraction of genomic DNA from whole blood and FFPE tissue. METHODS DNA was extracted from 10 whole blood and 10 FFPE specimens using six different commercial kits. RESULTS For whole blood, the mean DNA concentration obtained by Maxwell® 16 was significantly greater than either easyMAG® (p<0.0001) or QIAamp® Blood DNA kit (p<0.001). For FFPE, the mean DNA concentration obtained by the AllPrep® FFPE specific DNA/RNA kit was significantly greater than either the Maxwell® 16 (p<0.0001) or the general AllPrep® DNA/RNA kit (p<0.0001). CONCLUSIONS Comparative evaluation of the six DNA extraction kits indicated that the semi-automated Maxwell® 16 was superior for whole blood extraction while the manual AllPrep® FFPE DNA/RNA kit (Qiagen) performed better for FFPE DNA extraction in terms of quantity of DNA obtained. All six extraction methods (blood and FFPE) performed well in terms of purity. Although there were variances in the quantity of DNA obtained, there were no significant differences in the efficiency of these methods in yielding amplifiable DNA extracts, as demonstrated by β-actin for whole blood specimens. In evaluation of FFPE DNA extraction methods, the Qiagen AllPrep® FFPE DNA/RNA Mini Kit was the best for applications requiring larger amplicons, but for smaller amplicons the Maxwell was most consistent.
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A comparison of six methods for genomic DNA extraction suitable for PCR-based genotyping applications using ovine milk samples. Mol Cell Probes 2010; 24:93-8. [DOI: 10.1016/j.mcp.2009.11.001] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2009] [Revised: 10/01/2009] [Accepted: 11/02/2009] [Indexed: 11/19/2022]
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The era of molecular and other non-culture-based methods in diagnosis of sepsis. Clin Microbiol Rev 2010; 23:235-51. [PMID: 20065332 DOI: 10.1128/cmr.00043-09] [Citation(s) in RCA: 271] [Impact Index Per Article: 18.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Sepsis, a leading cause of morbidity and mortality throughout the world, is a clinical syndrome with signs and symptoms relating to an infectious event and the consequent important inflammatory response. From a clinical point of view, sepsis is a continuous process ranging from systemic inflammatory response syndrome (SIRS) to multiple-organ-dysfunction syndrome (MODS). Blood cultures are the current "gold standard" for diagnosis, and they are based on the detection of viable microorganisms present in blood. However, on some occasions, blood cultures have intrinsic limitations in terms of sensitivity and rapidity, and it is not expected that these drawbacks will be overcome by significant improvements in the near future. For these principal reasons, other approaches are therefore needed in association with blood culture to improve the overall diagnostic yield for septic patients. These considerations have represented the rationale for the development of highly sensitive and fast laboratory methods. This review addresses non-culture-based techniques for the diagnosis of sepsis, including molecular and other non-culture-based methods. In particular, the potential clinical role for the sensitive and rapid detection of bacterial and fungal DNA in the development of new diagnostic algorithms is discussed.
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Perandin F, Pollara PC, Gargiulo F, Bonfanti C, Manca N. Performance evaluation of the automated NucliSens easyMAG nucleic acid extraction platform in comparison with QIAamp Mini kit from clinical specimens. Diagn Microbiol Infect Dis 2009; 64:158-65. [PMID: 19500527 DOI: 10.1016/j.diagmicrobio.2009.02.013] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2008] [Revised: 02/16/2009] [Accepted: 02/19/2009] [Indexed: 11/30/2022]
Abstract
The performance of the NucliSens easyMAG platform for the extraction of nucleic acid from different clinical specimens was compared with a manual procedure. A total of 308 specimens were analyzed: 209 plasma samples collected for virus detection and quantification of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (n = 70), and 29 for HIV genotyping for drug resistance. Linearity of extraction was tested on dilution series of CMV and EBV; the correlation coefficient (R(2)) for standard curves based on repeated extraction runs was 0.99 for CMV and EBV. Inter- and intrarun variability was in accordance with previous studies, and the correlation between automated and manual extraction was very high. The concordant results were 95.7% for CMV and 100% for EBV. The results of sequence analysis for HIV drug resistance showed a concordance in 24 of 29 specimens. The NucliSens easyMAG is extremely easy to perform, is automated, and resulted in a strong reduction of hands-on time compared with manual protocol.
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Affiliation(s)
- Francesca Perandin
- Department of Experimental and Applied Medicine, University of Brescia, Italy.
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31
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Evaluation of new preanalysis sample treatment tools and DNA isolation protocols to improve bacterial pathogen detection in whole blood. J Clin Microbiol 2009; 47:2629-31. [PMID: 19535529 DOI: 10.1128/jcm.00821-09] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Two novel preanalysis sample treatment tools were evaluated in combination with four DNA extraction kits for the selective isolation of bacterial DNA from whole blood. The combination of performing a preanalysis sample treatment and using a larger sample volume increased the detection limit to 50 CFU per ml.
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32
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Loens K, Bergs K, Ursi D, Goossens H, Ieven M. Evaluation of NucliSens easyMAG for automated nucleic acid extraction from various clinical specimens. J Clin Microbiol 2006; 45:421-5. [PMID: 17166966 PMCID: PMC1829055 DOI: 10.1128/jcm.00894-06] [Citation(s) in RCA: 98] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The objectives of this study were to evaluate the performance of the NucliSens easyMAG platform for nucleic acid extraction from different clinical specimens compared to NucliSens miniMAG platform and manual QIAGEN extraction. The NucliSens easyMAG and the NucliSens miniMAG showed equal performance on 215 throat swabs since real-time nucleic acid sequence-based amplification scored the same samples positive for Mycoplasma pneumoniae (n=9) and Chlamydia pneumoniae (n=5) RNAs, although internal control RNA was slightly better detected with the NucliSens easyMAG (99.3% versus 96.8%). NucliSens easyMAG extracted nucleic acids more efficiently (higher recovery and/or fewer inhibitors) compared to QIAGEN extraction by showing, on average, lower Ct values in real-time LightCycler PCR, although 4 individual specimen out of 45 were found positive only with QIAGEN. For nine M. pneumoniae-positive throat swabs, the mean difference in Ct values between NucliSens easyMAG extraction and QIAGEN extraction was -2.26 (range, -5.77 to +0.60); for the detection of five C. pneumoniae-positive throat swabs, the average difference in Ct values between the two methods was -3.38 (range, -6.62 to -2.02); and for the detection of cytomegalovirus in 24 blood samples, the mean difference in Ct values between the two methods was -0.95 (range, -5.51 to +1.68). The NucliSens easyMAG is considerably easier to perform, efficiently extracts nucleic acids from throat swabs and whole blood, is automated, and has high throughput.
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Affiliation(s)
- K Loens
- Department of Medical Microbiology, University of Antwerp, Universiteitsplein 1 S009a, B-2610 Wilrijk, Belgium.
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Lu YQ, Han JX, Qi P, Xu W, Zu YH, Zhu B. Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA. World J Gastroenterol 2006; 12:7365-70. [PMID: 17143958 PMCID: PMC4087500 DOI: 10.3748/wjg.v12.i45.7365] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA.
METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified.
RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 104/mL, 6.3 × 102/mL and 1.6 × 103/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 109/mL, 2.08 × 106/mL and 4.40 × 107/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 105/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate.
CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.
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Affiliation(s)
- Yan-Qin Lu
- Shandong Medicinal Biotechnology Center, Shandong Academy of Medical Sciences, Key Laboratory of Ministry of Health for Biotech-Drugs, Jinan 250062, China
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Ulrich MP, Christensen DR, Coyne SR, Craw PD, Henchal EA, Sakai SH, Swenson D, Tholath J, Tsai J, Weir AF, Norwood DA. Evaluation of the Cepheid GeneXpert system for detecting Bacillus anthracis. J Appl Microbiol 2006; 100:1011-6. [PMID: 16630001 DOI: 10.1111/j.1365-2672.2006.02810.x] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
AIMS The Cepheid GeneXpert is a four-site, automated sample preparation and real-time PCR detection system. In this study, the capability of the GeneXpert to isolate and detect nucleic acid from Bacillus anthracis Ames spores was assessed. METHODS AND RESULTS A four-plex, dried-down bead cartridge containing PCR reagents specific for the pXO1 and pXO2 plasmids as well as sample processing and inhibition controls was evaluated. For B. anthracis Ames spores harbouring pXO1 and pXO2, samples containing 68 CFU per ml (148 spores per ml) were positive in all four replicates. A limited cross-reactivity panel, which included closely related Bacillus species, was also tested to determine the specificity of the pXO1 and pXO2 assays. No cross-reactivity occurred. Further, B. anthracis Sterne spore samples were analysed to compare results when processed using the GeneXpert to those run directly on the Cepheid SmartCycler without sample processing. The GeneXpert detection capability was three logs lower than the SmartCycler indicating the benefit of incorporating a nucleic acid extraction procedure. CONCLUSIONS This study demonstrates that the GeneXpert is a rapid and reliable system for simultaneously detecting the B. anthracis virulence plasmids pXO1 and pXO2. SIGNIFICANCE AND IMPACT OF THE STUDY The GeneXpert is the only platform currently available that is capable of both nucleic acid purification and real-time PCR detection enclosed within a single system. Further, all sample manipulations are automated, thus reducing errors associated with manual processing.
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Affiliation(s)
- M P Ulrich
- Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702-5011, USA
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Moriarity JR, Loftis AD, Dasch GA. High-throughput molecular testing of ticks using a liquid-handling robot. JOURNAL OF MEDICAL ENTOMOLOGY 2005; 42:1063-7. [PMID: 16465749 DOI: 10.1093/jmedent/42.6.1063] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/05/2023]
Abstract
To meet the need for high-throughput sample testing, DNA extraction kits based on the 96-well plate format have been developed for use with blood and tissue samples. These methods have not been applied to DNA extractions from ticks. To meet this need, we developed a high-throughput method for DNA extraction and polymerase chain reaction (PCR) testing of tick samples. A liquid-handling robot was used to extract DNA in a 96-well binding column plate with vacuum manifold. The quantity, purity, and quality of DNA recovered from Ixodes scapularis Say, 1821 nymphs with this method were reproducible and comparable with existing manual DNA extraction techniques. The DNA yield from pools of five nymphal ticks averaged 0.432 +/- 0.04 microg (95% CI). The robot also prepared real-time PCR reactions in 96-well plates, directly from the extracted DNA. A modification of the existing P20 tool resulted in accurate pipetting of 1- to 2-microl volumes with a reproducibility of +/- 0.038 microl when dispensing 1.0 microl. By using this process, 96 samples can be extracted and tested while reducing human labor to approximately 30 min.
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Affiliation(s)
- John R Moriarity
- Viral and Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
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Wilson D, Yen-Lieberman B, Reischl U, Warshawsky I, Procop GW. Comparison of five methods for extraction of Legionella pneumophila from respiratory specimens. J Clin Microbiol 2005; 42:5913-6. [PMID: 15583339 PMCID: PMC535306 DOI: 10.1128/jcm.42.12.5913-5916.2004] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The efficiencies of five commercially available nucleic acid extraction methods were evaluated for the recovery of a standardized inoculum of Legionella pneumophila in respiratory specimens (sputum and bronchoalveolar lavage [BAL] specimens). The concentrations of Legionella DNA recovered from sputa with the automated MagNA Pure (526,200 CFU/ml) and NucliSens (171,800 CFU/ml) extractors were greater than those recovered with the manual methods (i.e., Roche High Pure kit [133,900 CFU/ml], QIAamp DNA Mini kit [46,380 CFU/ml], and ViralXpress kit [13,635 CFU/ml]). The rank order was the same for extracts from BAL specimens, except that for this specimen type the QIAamp DNA Mini kit recovered more than the Roche High Pure kit.
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Affiliation(s)
- Deborah Wilson
- Section of Clinical Microbiology, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA
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Coyne SR, Craw PD, Norwood DA, Ulrich MP. Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit. J Clin Microbiol 2004; 42:4859-62. [PMID: 15472363 PMCID: PMC522347 DOI: 10.1128/jcm.42.10.4859-4862.2004] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Efficient, rapid, and reproducible procedures for isolating high-quality DNA before PCR gene amplification are essential for the diagnostic and molecular identification of pathogenic bacteria. This study evaluated the Qiagen QIAamp DNA Mini Kit and the Schleicher and Schuell IsoCode Stix DNA isolation device for isolating nucleic acid. Buffer, serum, and whole-blood samples were spiked with Bacillus anthracis Sterne vegetative cells and Yersinia pestis, while water was spiked with B. anthracis Sterne spores. Although minimal variations in limit of detection occurred among matrices, both the IsoCode Stix extraction method and the Qiagen procedure have comparable detection limits.
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Affiliation(s)
- Susan R Coyne
- Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702-5011, USA
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38
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Smith K, Diggle MA, Clarke SC. Automation of a fluorescence-based multiplex PCR for the laboratory confirmation of common bacterial pathogens. J Med Microbiol 2004; 53:115-117. [PMID: 14729931 DOI: 10.1099/jmm.0.05416-0] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
A fluorescence-based multiplex PCR was automated for the simultaneous detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae in clinical samples from patients with suspected meningitis. Sensitivity of one to two genome copies per 100 μl sample and specificity of 100 % for each organism were shown. Automation of DNA extraction, liquid handling, PCR and analysis are achieved on a single platform, which enables a high throughput and rapid turnaround of clinical samples that, in turn, leads to faster diagnosis. This is ultimately beneficial to the treatment of the patient and for public health management.
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Affiliation(s)
- Karen Smith
- Scottish Meningococcus and Pneumococcus Reference Laboratory, Stobhill Hospital, Glasgow, Scotland, UK 2Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, UK
| | - Mathew A Diggle
- Scottish Meningococcus and Pneumococcus Reference Laboratory, Stobhill Hospital, Glasgow, Scotland, UK 2Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, UK
| | - Stuart C Clarke
- Scottish Meningococcus and Pneumococcus Reference Laboratory, Stobhill Hospital, Glasgow, Scotland, UK 2Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, UK
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