猪细小病毒2型、3型、6型多重PCR检测方法的建立及初步应用

Development and application of multiplex PCR for the simultaneous detection of Porcine parvovirus 2,Porcine parvovirus 3,Porcine parvovirus 6

为建立同时快速检测猪细小病毒2型(PPV2)、猪细小病毒3型(PPV3)、猪细小病毒6型(PPV6)的方法,本研究根据PPV2、PPV3、PPV6各自的NS基因序列设计3对特异性引物,建立了一种能同时检测上述病原的多重PCR方法。结果显示,建立的多重PCR方法对PPV2、PPV3、PPV6的基因组DNA均能有效扩增,对猪圆环病毒2型(PCV2)、猪流行性腹泻病毒(PEDV)、猪繁殖与呼吸综合症病毒(PRRSV)、猪伪狂犬病毒(PRV)、副猪嗜血杆菌(HPS)、猪胸膜肺炎放线杆菌(APP)、日本乙型脑炎病毒(JEV)的核酸均无特异性扩增,特异性较强;对PPV2、PPV3、PPV6的重组质粒最低检测限分别为4.32×10^3拷贝/μL、9.62×10^3拷贝/μL、4.25×10^3拷贝/μL,与其他研究者已建立的单一PCR检测结果一致,敏感性较高。利用本研究建立的多重PCR方法对25份可疑临床样品检测,同时利用文献报道的单一PCR方法检测,二者结果一致。本研究建立的多重PCR方法为检测PPV,及该病的流行病学调查提供了技术支持。

In order to simultaneously and rapidly detect porcine parvovirus 2(PPV2),porcine parvovirus 3(PPV3),porcine parvovirus 6(PPV6),a multiplex PCR was developed based on three pairs of specific primers for each of viral NS gene.The results showed that this method was able to simultaneously amplify the specific fragments of PPV6,PPV2,PPV3,and no cross-reaction occurred among PCV2,PEDV,PRRSV,PRV,HPS,APP and JEV.Sensitivity test results showed that this method could amplify the three target genes with template amounts of 4.32×10^3 copies/μL,9.62×10^3 copies/μL,4.25×10^3 copies/μL for PPV2,PPV3,PPV6 respectively.This multiplex PCR method was applied to detect 25 clinical samples and the results were consistent with those of single PCR.The multiplex PCR assay developed in this study represents a useful tool to monitor the prevalence of PPV in China.