一步法CSFV和PRRSV双重实时荧光定量RT-PCR诊断方法的建立与初步应用

Development and application of double real-time fluorescent quantitative RT-PCR assay for detection of PRRSV and CSFV

根据猪瘟病毒(CSFV)和猪繁殖与呼吸综合征病毒(PRRSV)的序列,分别设计了1对特异性引物和1条Taq Man探针,建立了一步法检测CSFV和PRRSV双重实时荧光定量RT-PCR方法。结果表明,建立的方法特异性高,与PCV2,PRV,PPV,PEDV,JEV,BVDV无交叉反应;可以检出20个拷贝数的模板RNA,比常规RT-PCR方法的灵敏度高10倍;对同一样品进行4次重复试验,变异系数(CV)低于4%,方法重复性好。应用建立的荧光定量RT-PCR和常规RT-PCR对50份疑似病例组织进行检测,结果荧光定量RT-PCR检出30份PRRSV阳性,13份CSFV阳性,均高于常规RT-PCR方法的检出数(24份PRRSV阳性,10份CSFV阳性)。本研究建立的双重实时荧光定量RT-PCR方法具有快速、特异性好、灵敏度高的特点,可用于PRRSV和CSFV的实验室快速诊断和流行病学调查。

To identify classical swine fever virus（ CSFV） and porcine reproductive and respiratory syndrome virus（ PRRSV） rapidly,a double real-time fluorescent RT-PCR assay of CSFV and PRRSV was designed,along with primers and Taq Man probes based on CSFV and PRRSV genome sequences. It was shown that the proposed assay proved to be specific,as no amplification was found of PCV2,PRV,PPV,PEDV,JEV,BVDV. The assay was sensitive to as low as 20 copies of template RNA of PRRSV and CSFV,and the sensitivity was 10 times higher than that of the conventional RT-PCR. The coefficient variation（ CV%） of intra / inter-assay for the same RNA sample was less than 4%. Fifty samples were examined by the proposed fluorescent quantitative RT-PCR and the conventional RT-PCR,respectively,and there were 30 and 13 samples found to be infected with PRRSV and CSFV,respectively,by the proposed assay,but only 24 and 10 samples were found to be infected with PRRSV and CSFV,respectively,by the conventional RT-PCR. These results showed that the developed double real-time fluorescent quantitative RTPCR assay was rapid,specific,sensitive and simple for the detection of PRRSV and CSFV,which could be used as a rapid detection method for the epidemiologic survey of PRRSV and CSFV.