Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus

Bok Soon Min, Yoon Ju Noh, Jin Ho Shin, Sun Young Baek, Kyung Il Min, Seung Rel Ryu, Byoung Guk Kim, Mi Kyung Park, Seung Eun Choi, Eun Hee Yang, Sue Nie Park, Sook Jin Hur, Byung-Yoon Ahn

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Abstract

Nucleic acid amplification techniques are used frequently for rapid diagnosis of viral diseases. In this study, a real-time polymerase chain reaction protocol that uses primers specific for the viral VP4 gene and the commercial SYBR Green reagent were evaluated for the quantitative measurement of human rotavirus (HRV) RNA in human stool specimens. SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. The assay resulted in a sensitive and reproducible detection of targets ranging from low (<102 rotavirus cDNA copies/reaction) to high numbers (>106 rotavirus cDNA copies/reaction). No cross-reaction was found with crude cell culture stocks of coxsackievirus, echovirus, poliovirus, hepatitis A virus and adenovirus. Analysis with the HRV cDNA standard demonstrated high reproducibility with a coefficient of variation (CV) of 0.2-0.9%. Daily performance among three different laboratories showed a CV no greater than 8%, indicating an intermediate level of variation. These results demonstrate the feasibility of this method for quantitative analysis of human rotavirus in clinical samples.

Original languageEnglish
Pages (from-to)280-286
Number of pages7
JournalJournal of Virological Methods
Volume137
Issue number2
DOIs
Publication statusPublished - 2006 Nov 1

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Keywords

  • Human rotavirus
  • Quantitative assay
  • Real-time PCR

ASJC Scopus subject areas

  • Virology

Cite this

Min, B. S., Noh, Y. J., Shin, J. H., Baek, S. Y., Min, K. I., Ryu, S. R., Kim, B. G., Park, M. K., Choi, S. E., Yang, E. H., Park, S. N., Hur, S. J., & Ahn, B-Y. (2006). Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus. Journal of Virological Methods, 137(2), 280-286. https://doi.org/10.1016/j.jviromet.2006.06.028