Development of a TaqMan--based Real--time reverse transcription PCR for detecting genogroup II norovirus in clinical stool samples

Norovirus has been recognized as one of the major causes of gastroenteritis in the world.In order to control its epidemic,we designed the specific primers and probe following large scale norovirus genome consensus analysis and subsequently established a TaqMan-based Real-time RT-PCR assay for detection of genogroupⅡ(GGⅡ) norovirus RNA.The results showed that the assay developed in this paper possessed high specificity for norovirus nucleic acid detection and without any evident cross-reaction with other viruses,including rotavirus,adenovirus and hepatitis A virus.The sensitivity of the assay was as low as 10~2 copies per reaction.The assay was linear within 8-log dynamic range between 10~9 and 10~2 copies.The correlation coefficient of the standard curve was-1.00.The coefficients of variation(CV) of the Ct values of the standard plasmid were 0.28%-1.63%(n=6)in intraassay and 0.28%-1.05%(n=3)in inter-assay,respectively,while the CV of the same sample in 6 RNA extractions and reverse transcriptions was 3.88%(n=6).The detection rate of the assay was a little higher but not statistically different from that of the conventional RT-PCR assay by detecting 212 stool samples.Taken together,the Real-time PCR assay successfully developed in the present study revealed its potential for norovirus diagnosis in clinical stool samples.