小反刍兽疫疫苗毒株与强毒株的鉴别性荧光RT-PCR检测方法的建立

摘要

Through the comparison of the N gene of PRRV from different countries and areas in GenBank,three pairs of primers and three different fluorescent-labeled probes were designed,and a duplex RT-qPCR assay was established based on a conservative sequence of PPRV and a specific sequence of the vaccine strain,meanwhile a specific qPCR assay was built based on a specific sequence of the virulent wild strain. Sensitivity test showed that this duplex assay could detect 101 TCID50/mL in HEX channel and 102 TCID50/mL in TAMRA channel,enough for the diagnosis of PPRV. The specificity test showed that it could react only with PRRV and not with the other similar viruses. 14 sheep serum samples collected from Tibet areas were detected using the assay,and the results indicated that this method was specific and sensitive,it could also distinct the vaccine strain and virulent wild strain of PPRV.%　　对GenBank已公布的小反刍兽疫病毒N基因序列进行分析,设计引物与探针,建立PPRV通用型与Ⅱ系疫苗毒特异型二重实时荧光RT-PCR方法,同时建立针对PPRVⅣ系强毒株的特异型荧光PCR方法。特异性试验和灵敏度试验表明：建立的二重荧光RT-PCR方法可特异性检测PPRV病毒,其HEX信号通道(通用型)和TAMRA信号通道(Ⅱ系疫苗毒特异型)的检测灵敏度分别可达101 TCID50/mL和102 TCID50/mL,完全满足PPRV的检测要求。用二重荧光RT-PCR方法对西藏采集的14份羊血清样品进行检测,并用建立的Ⅳ系强毒株特异型荧光PCR方法对二重荧光RT-PCR的检测效果进行评估,结果显示,该方法可有效甄别PPRV强毒株和疫苗毒株,避免假阳性结果的出现。