猪6种常见病毒多重PCR检测方法的建立及应用

旨在建立一种可同时检测猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)、猪细小病毒(PPV)和猪巨细胞病毒(PCMV)的多重PCR检测方法。参考相关文献及序列比对结果设计6对特异性引物,建立了可同时检测以上6种病毒的多重PCR方法并应用此方法对临床病例进行了检测。结果表明,所建立的多重PCR方法灵敏度高,对6种病毒的最低核酸检测量分别为12.8pg(PRRSV)、46pg(CSFV)、16pg(PRV)、23.5pg(PCV-2)、72pg(PPV)、8.6pg(PCMV),对猪流感病毒(SIV)、猪乙型脑炎病毒(JEV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV)无特异性扩增。应用该方法对临床145份样品进行检测,总阳性率为83.45%,2种以上病毒混合感染阳性率为69.66%。说明所建立的多重PCR检测方法敏感,可用于猪群中上述6种猪病病原的单一感染或混合感染的鉴别诊断。

Establishment and Application of Multiplex PCR for Detecting Six Kinds of Swine Viruses

The aim of this assay was to establish a multiplex PCR method for simultaneously detecting por-cine reproductive and respiratory syndrome virus (PRRSV),classical swine fever virus (CSFV),porcine pseudorabies virus (PPV),porcine circovirus type 2 (PCV-2),porcine parvovirus (PPV)and porcine cyto-megalovirus (PCMV).Six pairs of specific primers were designed for a multiplex PCR based on the conser-vative nucleotides according to references and sequence analysis.Samples collected from clinically ill pigs were detected by the multiplex PCR method.The results of sensitivity and specificity tests showed that the minimum amounts of nucleic acid detection by the mPCR were 12.8,46,16,23.5,72,8.6 pg respectively, and the amplification results of swine influenza virus (SIV),Japanese encephalitis virus (JEV),porcine epi-demic diarrhea virus,(PEDV),transmissible gastroenteritis virus (TGEV)and porcine rotavirus (PoRV) were all negative.A total of 145 specimens piglets were tested by the multiplex PCR method,the total pos-itive rate was 83.45%,and the co-infection rate was 69.66%.We successfully established a multiplex PCR method with high sensitivity,and specificity and the method was well used in clinical tests.