绵羊痘病毒、山羊痘病毒及羊口疮病毒多重PCR检测方法的建立和应用

为了建立鉴别绵羊痘病毒(SPPV)、山羊痘病毒(GTPV)和羊口疮病毒(ORFV)的多重PCR检测方法,针对GenBank中3种病毒的基因组序列,合成了3对引物,通过优化多重PCR反应条件,建立了鉴别检测3种病毒的多重PCR方法。特异性试验表明,应用该方法可分别扩增出3种病毒对应的目的片段,对大肠埃希菌、沙门菌、猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、Vero细胞、正常羊组织的DNA和灭菌双蒸水均无扩增;敏感性试验表明,该方法最低检测量分别为30.46pg/μL的绵羊痘病毒、28.9pg/μL的山羊痘病毒和26.94pg/μL的羊口疮病毒基因组DNA;应用本方法对85份临床病料进行检测,结果与其他已建立的单项PCR检测方法结果一致,说明该方法可以用于临床上SPPV、GTPV和ORFV的鉴别诊断。

Establishment and Application of Multiplex PCR for Detecting Sheeppox Virus,Goatpox Virus and Orf Virus

A multiplex polymerase chain reaction assay was developed for the detection of sheeppox virus,goatpox virus and orf virus.According to the genome sequence of SPPV,GTPV and ORFV in GenBank,three pairs of primers were synthetized.The multiplex PCR reaction condition was optimized,and the multiplex PCR for simultaneously detecting SPPV,GTPV and ORFV was established.The specificity test showed that target fragments were obtained from the genomic DNA of three virus respectively.Three fragments were amplified from the mixed DNA sample of SPPV, GTPV and ORFV simultaneously,and no amplification was obtained from Escherichia coli,Salmonella, PCV2,PPV,Vero cell line,healthy goat or sheep tissue, and ddH2O.The sensitivity test showed that the multiplex PCR could detect 30.46 pg/μL of SPPV DNA,28.9 pg/μL of GTPV DNA and 26.94 pg/μL of ORFV DNA.The results of multiplex PCR were consistent with the established PCR detection method in testing 85 clinical samples.These results indicated that multiplex PCR has values for detecting of SPPV,GTPV and ORFV in clinic.