猪瘟病毒野生株和疫苗株荧光RT-PCR鉴别方法的建立

Development of Real-time RT-PCR using MGB Fluorescence Probe for Differentiation of Wild-type and Vaccine Strains of Classical Swine Fever Virus

摘要:

为了建立猪瘟病毒野生株和疫苗株荧光RT-PCR鉴别方法，设计了1对通用引物和2条特异性MGB探针。此外，为防止假阴性结果出现，制备了假病毒用于全程检测监控。测试了本方法的特异性，灵敏度，重复性等技术指标。结果显示，在选定的猪病相关病毒组内特异性符合率达100%；对野毒株和疫苗株的检测灵敏度分别达到1 TCID50/mL和0.1 TCID50/mL；重复性实验表明组内和组间变异系数均在0.7~2.2%之间。临床比对试验结果显示，荧光RT-PCR对猪肉、脾脏和血液病料进行猪瘟野毒检测的阳性率分别为66.7%、60.0%、77.8%，而传统方法的阳性率分别为52.4%、40.0%、50.0%；检出率比传统方法要高。此外，临床试验样本中，PCR抑制物在猪肉、脾脏和血液病料中存在的比例分别为9.5%、10.0%和5.6%，显示了假病毒作为内标对PCR检测监控的重要作用。

Abstract:

In order to the develop multiples real-time PCR for detection and differentiation of wild-type and vaccine strains of classical swine fever virus, a pair of common primers and two differently-labeled specific MGB Fluorescence probes were designed against conserved domain of the 5’-UTR of wild-type viruses and C-strain vaccine. Moreover, in order to prevent the false negative result, pseudovirus was prepared for monitoring. The multiples real-time PCR method showed 100% specificity for the selected panel with the sensitivity being of 1 TCID50/mL for wild-type virus and 0.1 TCID 50/mL for C-strain vaccine. The reproducibility experiments showed that the variation coefficients (%CV) of intra-assay and inter-assay ranged from 0.7% to 2.2%. By using this method, CSFV RNA was detected in specimens of meat, spleen and blood with positive rates of 66.7%, 60.0% and 77.8%，respectively, while the positive rates obtained by using the standard assay was 52.4%, 40.0% and 50.0%, respectively. The result statistics showed the existence of PCR inhibitos in the clinical specimens and specimens with inhibition ratio being of 5.6~10.0%, indicating the importance of pseudovirus as IAC when PCR was used to detect the RNA of wild-type viruses and C-strain vaccine.