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

Research output: Contribution to journalArticle

32 Citations (Scopus)

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

Fingerprint

Rotavirus
Real-Time Polymerase Chain Reaction
Complementary DNA
Nucleic Acid Amplification Techniques
Human Enterovirus B
Hepatitis A virus
Temperature
Poliovirus
Viral Genes
Enterovirus
Cross Reactions
Virus Diseases
Adenoviridae
Freezing
Cell Culture Techniques
Fluorescence
RNA
Polymerase Chain Reaction

Keywords

  • Human rotavirus
  • Quantitative assay
  • Real-time PCR

ASJC Scopus subject areas

  • Virology

Cite this

Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus. / Min, Bok Soon; Noh, Yoon Ju; Shin, Jin Ho; Baek, Sun Young; Min, Kyung Il; Ryu, Seung Rel; Kim, Byoung Guk; Park, Mi Kyung; Choi, Seung Eun; Yang, Eun Hee; Park, Sue Nie; Hur, Sook Jin; Ahn, Byung-Yoon.

In: Journal of Virological Methods, Vol. 137, No. 2, 01.11.2006, p. 280-286.

Research output: Contribution to journalArticle

Min, BS, Noh, YJ, Shin, JH, Baek, SY, Min, KI, Ryu, SR, Kim, BG, Park, MK, Choi, SE, Yang, EH, Park, SN, Hur, SJ & 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, vol. 137, no. 2, pp. 280-286. https://doi.org/10.1016/j.jviromet.2006.06.028
Min, Bok Soon ; Noh, Yoon Ju ; Shin, Jin Ho ; Baek, Sun Young ; Min, Kyung Il ; Ryu, Seung Rel ; Kim, Byoung Guk ; Park, Mi Kyung ; Choi, Seung Eun ; Yang, Eun Hee ; Park, Sue Nie ; Hur, Sook Jin ; Ahn, Byung-Yoon. / Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus. In: Journal of Virological Methods. 2006 ; Vol. 137, No. 2. pp. 280-286.
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