Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus.
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 (<10(2)rotavirus cDNA copies/reaction) to high numbers (>10(6)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.
Min
Bok-Soon
BS
Department of Biologics Evaluation, Korea Food and Drug Administration, Republic of Korea.
Noh
Yoon-Ju
YJ
Shin
Jin-Ho
JH
Baek
Sun-Young
SY
Min
Kyung-Il
KI
Ryu
Seung-Rel
SR
Kim
Byoung-Guk
BG
Park
Mi-Kyung
MK
Choi
Seung-Eun
SE
Yang
Eun-Hee
EH
Park
Sue-Nie
SN
Hur
Sook-Jin
SJ
Ahn
Byung-Yoon
BY
D000704:Analysis of Variance; D052160:Benzothiazoles; D036022:Capsid Proteins / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D003959:Diamines; D005243:Feces / Q000821:virology; D006801:Humans; D007223:Infant; D015588:Observer Variation; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis / Q000821:virology*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D052160:Benzothiazoles; D036022:Capsid Proteins; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C060339:VP4 protein, Rotavirus; C098022:SYBR Green I