猪轮状病毒TaqMan荧光定量检测方法的建立及初步应用

Establishment and preliminary application of TaqMan fluorescence quantitative detection method for porcine rotavirus

为了对早期猪轮状病毒(porcine rotavirus,PoRV)感染情况进行监测,根据GenBank中已有的A型PoRV VP6序列,设计2对引物和荧光定量探针,建立了TaqMan荧光定量RT-PCR方法,并对该方法特异性、灵敏性和重复性进行评价。结果显示所建立的TaqMan荧光定量RT-PCR方法特异性强、重复性好、灵敏度高,可检测到101拷贝/μL。经对2366份腹泻仔猪的粪便样品进行检测,所建立的方法检出率(5.92%,140/2366)高于常规RT-PCR(4.95%,117/2366),两者的符合率为98.58%。结果表明,本研究建立的PoRV TaqMan荧光定量RT-PCR方法可用于猪轮状病毒病的临床检测。

In order to monitor the early porcine rotavirus infection,according to the existing porcine rotavirus A VP6 sequence in GenBank,two pairs of primers and fluorescence probes were designed,and the TaqMan real-time RT-PCR method was established,and the specificity,sensitivity and repeatability of the method were evaluated.The results showed that the established TaqMan real-time RT-PCR method had strong specificity,good repeatability and high sensitivity,and could detect 10~1 copies/μL.After testing the fecal samples of 2366 diarrheal piglets,the detection rate of the established method(5.92%,140/2366)was higher than that of conventional RT-PCR(4.95%,117/2366),and the compliance rate of the two was 98.58%.The results showed that the TaqMan fluorescence RT-PCR method established in this study can be used for the clinical detection of porcine rotavirus disease.