血清型特异性水泡性口炎病毒同步检测和鉴别方法的建立

Establishment of a Duplex Real-time RT-PCR for Detection and Identification of Serotype-specific VSV Strains

为准确地鉴别诊断新泽西型水泡性口炎病毒(VSV-NJ)和印第安纳型水泡性口炎病毒(VSV-IND),本研究建立了可以同时检测和鉴别诊断VSV-NJ和VSV-IND的双重荧光RT-PCR方法,并对方法性能进行了测试和初步应用。结果显示,建立的双重荧光RT-PCR特异性为100%,最低检测限1~10个拷贝/μL,扩增效率E值均在99%左右,R2值分别为0.999和0.998。VSV-NJ和VSV-IND单荧光RT-PCR与双重荧光RT-PCR的相关性系数分别为0.9997和0.9994,与单荧光RT-PCR相比,该引物和探针的双重体系混合对特异性和敏感性均未产生明显干扰。用已知VSV-NJ及VSV-IND阳性核酸验证,发现符合率为100%,对50份临床样品的检测均为阴性,与单荧光RT-PCR结果一致。本研究建立的双重荧光RT-PCR方法实现了VSV-NJ和VSV-IND的同步检测和鉴别,为水泡性口炎的防控提供了技术储备。

In order to accurately identify and differentiate the serotype-specific strains of New Jersey type of vesicular stomatitis virus(VSV-NJ)and Indiana type of vesicular stomatitis virus(VSV-IND),a duplex real-time RT-PCR method was established for simultaneous detection of the two serotypes of viruses,and its performance was tested and initial application was carried out.The results showed that the specificity of the established method was 100%,with the lowest detection limit of 1~10 copies/μL,both of the amplification efficiencies(E)were about 99%,and R2 values were 0.999 and 0.998,respectively.The correlation coefficients between singular real-time RT-PCR and duplex realtime RT-PCR for VSV-NJ and VSV-IND were 0.9997 and 0.9994,respectively.Compared to singular real-time RTPCR,both the specificity and sensitivity were not affected by the mixture of the primers and probes.The coincidence rate was 100%as verified by the known positive nucleic acids of VSV-NJ and VSV-IND,and 50 clinical samples were tested to be negative,which was consistent with the results by singular real-time RT-PCR.As a conclusion,the method established in this study could simultaneously detect and differentiate VSV-NJ and VSV-IND,which would provide technical reserves for prevention and control of VSV.