猪细小、猪伪狂犬和猪繁殖与呼吸综合征病毒三重RT-qPCR检测方法的建立及初步应用

Establishment and Preliminary Application of Triple RT-qPCR Detection Method for Porcine Parvovirus,Porcine Pseudorabies Virus,and Porcine Reproductive and Respiratory Syndrome Virus

针对猪细小(PPV)、猪伪狂犬(PRV)和猪繁殖与呼吸综合征(PRRSV)病毒的保守区域设计用于构建荧光定量PCR体系的引物和探针.以构建的重组质粒标准品为模板建立三重荧光定量PCR体系,并对三重荧光定量PCR体系进行优化并进行特异性、灵敏性、重复性等试验.最后利用构建的三重荧光定量PCR体系和普通PCR体系分别检测病料以进行比较.结果表明:三种病毒在单重和三重荧光定量PCR体系中均无非特异性扩增;三种病毒在单重和三重荧光定量PCR体系中的灵敏度均可达到10 copies/μL;单重和三重荧光定量PCR体系组内和组间重复性变异系数均在5%以内;单重和三重荧光定量PCR在稀释度范围内呈现良好的线性关系.应用本研究构建的三重荧光定量PCR体系对164份临床组织样品进行检测,其中PPV阳性检出率为16.7%,PRV阳性检出率为10%,PRRSV阳性检出率为18.9%;普通PCR检测中PPV阳性检出率为6.5%,PRV阳性检出率为3.6%,PRRSV阳性检出率为7.5%.说明本研究建立的PPV、PRV和PRRSV三重荧光定量PCR检测方法符合临床检测要求并且比普通PCR方法更敏感、更特异.

Primers and probes for constructing a fluorescent quantitative PCR system were designed for the conserved regions of porcine parvovirus(PPV),porcine pseudorabies virus(PRV),and porcine reproductive and respiratory syndrome virus(PRRSV).The triple fluorescence quantitative PCR system was established with the constructed recombinant plasmid standard product as the template,and the triple fluorescence quantitative PCR system was optimized and tested for specificity,sensitivity,and repeatability.Finally,the constructed triple fluorescence quantitative PCR system and ordinary PCR system were used for clinical testing,and then the results were compared.The results showed that:Three viruses had no non-specific amplification in single and triple fluorescence quantitative PCR systems;The sensitivity of the three viruses in single and triple fluorescent quantitative PCR systems could reach 10 copies/μL;The repeatability coefficients of variation within and between groups in single and triple fluorescent quantitative PCR systems were within 5%;Single and triple fluorescence quantitative PCR showed a good linear relationship within the dilution range.The triple fluorescence quantitative PCR system constructed in this study was used to detect 164 clinical tissue samples.The positive detection rate of PPV was 16.7%,and the positive detection rate of PRV was 10%,and the positive detection rate of PRRSV was 18.9%.In the ordinary PCR test,the positive detection rate of PPV was 6.5%,and the positive detection rate of PRV was 3.6%,and the positive detection rate of PRRSVV was 7.5%.The results indicate that the triple fluorescence quantitative PCR method for PPV,PRV,and PRRSV established in this study meets the clinical requirements,and it is more sensitive and specific than the ordinary PCR method.