牛星状病毒和诺如病毒双重实时荧光定量RT-PCR检测方法的建立及应用OA

[Objective]The rapid method for simultaneous detection of BAstV and BNoV was established to provide guidance for timely formulating prevention and control measures against relative viral diseases.[Method]The specific primers were designed according to the conserved regions of ORF2 gene in BAstV and RdRp gene in BNoV respectively,and a duplex real-time fluorescence quantitative RT-PCR method for simultaneous detection of BAstV and BNoV was established by using a portable fluorescence quantitative RCR instrument.The sensitivity,specificity and repeatability of the established method were tested.The clinical samples were detected by the established method.[Result]The established duplex TaqMan probe fluorescence quantitative RCR method with a strong specificity could amplify the nucleic acids of BAstV and BNoV,but could not amplify the nucleic acids of common bovine viral diarrhea(BVDV),bovine cyclic curvature virus(BToV)and bovine coronavirus(BCoV).The minimum detection limit of BAstV and BNoV detected by the established duplex TaqMan probe fluorescence quantitative RCR method with a higher sensitivity reached 1.19 pg/µL and 0.82 pg/µL respectively.The variable coefficient of the established duplex TaqMan probe fluorescence quantitative RCR method with a good repeatability was less than 1%in the repeatability tests.The positive detection rate of BAstV and BNoV from 213 clinical samples was 23.94%and 7.98%respectively,and significantly higher than that of single SYBR Green dye method.[Conclusion]The established duplex fluorescence PCR method with the higher sensibility and strong specificity for detection of BAstV and BNoV can complete detection of BAstV and BNoV within 60 minutes by using a portable fluorescence quantitative PCR instrument and be applied in rapid detection of BAstV and BNoV infection in livestock farms.