检测PRRSV及鉴别HP-PRRSV毒株的二重PCR与二重TaqMan荧光定量PCR方法OA北大核心CSTPCD

In order to achieve early detection of porcine reproductive and respiratory syndrome virus(PRRSV)and rapid identification of highly pathogenic porcine reproductive and respiratory syn-drome virus(HP-PRRSV)variants,two pairs of specific primers were designed based on characteristics of amino acid deletion at positions 481 and 532-560 of HP-PRRSV Nsp2 protein,a duplex PCR detection method was established by optimizing the reaction conditions.The specific primers and probes were de-signed based on PRRSV M gene and HP-PRRSV Nsp2 gene,a duplex TaqMan real-time PCR method was estab-lished by optimizing the reaction conditions.The sensitivity,specificity,and reproducibility of both methods were evaluated.The sensitivity test results showed that the lowest detection limits of the duplex PCR method were 0.058 ng/μL(HP-PRRSV)and 4.7 ng/μL(HP-PRRSV);the minimum detection limits of the duplex TaqMan real-time PCR method were 10 copies/μL(PRRSV)and 100 copies/μL(HP-PRRSV),and there was no cross-reactivity between these two detection methods and other swine pathogens,and the repeatability test results were good.A total of 131 clinical samples detection showed positive rate of PRRSV was 12.2％(16/131),the positive rate of HP-PRRSV was 17.6％(23/131),and mixed infection rate was 8.4％(11/131).It was confirmed that the duplex PCR and duplex TaqMan real-time PCR detection methods were successfully established,which could provide technical support for the prevention and control of porcine reproductive and respiratory syndrome in clinical practice.