Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Virology. Nov 12, 2015; 4(4): 365-371
Published online Nov 12, 2015. doi: 10.5501/wjv.v4.i4.365
Rapid genotyping of human rotavirus using SYBR green real-time reverse transcription-polymerase chain reaction with melting curve analysis
Yupin Tong, Bonita E Lee, Xiaoli L Pang
Yupin Tong, Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2J2, Canada
Bonita E Lee, Department of Pediatrics, University of Alberta, Edmonton, Alberta T6G 2J2, Canada
Xiaoli L Pang, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta T6G 2J2, Canada
Xiaoli L Pang, Provincial Laboratory for Public Health, University of Alberta Hospital, Edmonton, Alberta T6G 2J2, Canada
Author contributions: Tong Y performed the experiments, analyzed the data and wrote the manuscript; Lee BE contributed data and sample selections for the study and revised the manuscript; Pang XL designed the research and revised the manuscript.
Institutional review board statement: The study was reviewed and approved by the Provincial Laboratory for Public Health Edmonton Alberta (Provlab) Institutional Review Board.
Conflict-of-interest statement: None.
Data sharing statement: Technical appendix, clinical dataset is available from the corresponding author at xiao-li.pang@albertahealthservices.ca.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Xiaoli L Pang, PhD, Provincial Laboratory for Public Health, University of Alberta Hospital, WMC 2B4.58, 8440 112 Street, Edmonton, Alberta T6G 2J2, Canada. xiao-li.pang@albertahealthservices.ca
Telephone: +1-780-4073483 Fax: +1-780-4078984
Received: May 29, 2015
Peer-review started: June 2, 2015
First decision: June 18, 2015
Revised: August 1, 2015
Accepted: September 29, 2015
Article in press: September 30, 2015
Published online: November 12, 2015
Processing time: 167 Days and 13.7 Hours
Abstract

AIM: To develop a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay to genotype rotavirus (G and P) in Alberta from January 2012 to June 2013.

METHODS: We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR (rt-gPCR) by selecting genotype-specific primers of published conventional RT nested PCR (cnRT-PCR) assay and optimizing the amplification conditions. cDNA was first synthesized from total RNA with SuperScript™ II reverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR. After the PCR reaction, melting curve analysis was used to determine specific genotype. Sixteen samples previously genotyped using cnRT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis. Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay. The amplicon size of each available genotype was determined by gel-electrophoresis and DNA sequences were obtained using Sanger-sequencing method. After validation and optimization, the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January 2012 and June 2013.

RESULTS: The new rt-gPCR assay was validated and optimized. The assay detected G1 to G4, G9, G12 and P[4] and P[8] that were available as positive controls in our laboratory. A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80 °C to 82 °C. The sensitivity of rt-gPCR was comparable to cnRT-PCR with 100% correlation of the 16 samples with known G and P genotypes. No cross reaction was found with other gastroenteritis viruses. Using the new rt-gPCR assay, genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus: G1P[8] (42.6%), G2P[4] (4.9%), G3P[8] (10.7%), G9P[8] (10.7%), G9P[4] (6.6%), G12P[8] (23.0%), and unknown GP[8] (0.8%). For the first time, G12 rotavirus strains were found in Alberta and G12 was the second most common genotype during the study period. Gel electrophoresis of all the genotypes showed expected amplicon size for each genotype. The sequence data of the two G12 samples along with other genotypes were blasted in NCBI BLAST or analyzed with Rota C Genotyping tool (http://rotac.regatools.be/). All genotyping results were confirmed to be correct.

CONCLUSION: rt-gPCR is a useful tool for the genotyping and characterization of rotavirus. Monitoring of rotavirus genotypes is important for the identification of emerging strains and ongoing evaluation of rotavirus vaccination programs.

Keywords: Rotavirus A; Melting temperature; Real-time polymerase chain reaction; SYBR green; Genotyping

Core tip: Genotyping rotavirus is essential for monitoring strain shifts in rotavirus surveillance and vaccine evaluation. Current conventional semi-nested real-time reverse transcription-polymerase chain reaction (RT-PCR), the most commonly used rotavirus genotyping assay is a labor-intensive, complex multi-step procedure and has long turn around-time. The newly developed SYBR Green real time RT-PCR assay is simple, fast and has comparable sensitivity and specificity as conventional semi-nested RT-PCR. This new assay was used to genotype clinical samples which tested positive for rotavirus from January 2012 to June 2013 and new emerging G12 strains were identified in Alberta, Canada.