The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting rotavirus A and norovirus. Dung, T. T. N., Phat, V. V., Nga, T. V. T., My, P. V. T., Duy, P. T., Campbell, J. I., Thuy, C. T., Hoang, N. V. M., Van Minh, P., Le Phuc, H., Tuyet, P. T. N., Vinh, H., Kien, D. T. H., Huy, H. L. A., Vinh, N. T., Nga, T. T. T., Hau, N. T. T., Chinh, N. T., Thuong, T. C., Tuan, H. M., Simmons, C., Farrar, J. J., & Baker, S. Journal of virological methods, 187(1):138–143, January, 2013. doi abstract bibtex Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits.
@article{dung_validation_2013,
title = {The validation and utility of a quantitative one-step multiplex {RT} real-time {PCR} targeting rotavirus {A} and norovirus.},
volume = {187},
copyright = {Copyright (c) 2012 Elsevier B.V. All rights reserved.},
issn = {1879-0984 0166-0934},
doi = {10.1016/j.jviromet.2012.09.021},
abstract = {Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10\% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23\% more NoV genogroup II positive samples from individuals with diarrhea and 9\% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits.},
language = {eng},
number = {1},
journal = {Journal of virological methods},
author = {Dung, Tran Thi Ngoc and Phat, Voong Vinh and Nga, Tran Vu Thieu and My, Phan Vu Tra and Duy, Pham Thanh and Campbell, James I. and Thuy, Cao Thu and Hoang, Nguyen Van Minh and Van Minh, Pham and Le Phuc, Hoang and Tuyet, Pham Thi Ngoc and Vinh, Ha and Kien, Duong Thi Hue and Huy, Huynh Le Anh and Vinh, Nguyen Thanh and Nga, Tran Thi Thu and Hau, Nguyen Thi Thu and Chinh, Nguyen Tran and Thuong, Tang Chi and Tuan, Ha Manh and Simmons, Cameron and Farrar, Jeremy J. and Baker, Stephen},
month = jan,
year = {2013},
pmid = {23046990},
pmcid = {PMC3528950},
keywords = {*Multiplex Polymerase Chain Reaction, *Real-Time Polymerase Chain Reaction, Caliciviridae Infections/*diagnosis/virology, Child, Preschool, Diarrhea/diagnosis/virology, Feces/virology, Gastroenteritis/diagnosis/genetics, Genotype, Humans, Infant, Infant, Newborn, Norovirus/genetics/*isolation \& purification, RNA, Viral/analysis, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, Rotavirus Infections/*diagnosis/virology, Rotavirus/genetics/*isolation \& purification},
pages = {138--143},
}
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J.","Baker, S."],"year":2013,"bibtype":"article","biburl":"https://api.zotero.org/users/4242995/collections/GEVPECXU/items?key=zD4uWY4xk1YOLS3UQ7bLDMqH&format=bibtex&limit=100","bibdata":{"bibtype":"article","type":"article","title":"The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting rotavirus A and norovirus.","volume":"187","copyright":"Copyright (c) 2012 Elsevier B.V. All rights reserved.","issn":"1879-0984 0166-0934","doi":"10.1016/j.jviromet.2012.09.021","abstract":"Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. 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