ASFV和PRV野毒及PRV疫苗毒多重纳米PCR检测方法的建立及应用

Establishment and application of multiplex nano-PCR detection method for ASFV,PRV wild virus and PRV vaccine virus

为建立一种可同时检测ASFV、PRV野毒和PRV疫苗毒(缺失g E基因)的方法,本研究以ASFV的B646L基因、PRV的g E基因以及PRV的g D基因为检测对象,建立了可同时检测ASFV、PRV野毒和PRV疫苗毒的多重纳米PCR方法,并对其特异性、敏感性进行了研究。结果显示,该方法可特异性扩增ASFV、PRV野毒和PRV疫苗毒的目的基因,而检测塞内卡病毒、猪血凝性脑脊髓炎病毒、猪水疱性口炎病毒、猪圆环病毒2型时结果均呈阴性;B646L基因、g D基因和g E基因的检测限分别为4、7和8 copies/μL。此外,将商品化试剂盒检验结果与本试验建立的多重纳米PCR检测结果进行比对,结果表明符合率为100%。综上所述,本试验建立的多重纳米PCR方法具有特异性好、灵敏度高的优点,且能够同时检测ASFV、PRV野毒和PRV疫苗毒,可用于猪重大疾病的鉴别诊断和疫病防控。

In order to establish a method for simultaneous detection of ASFV,PRV wild virus and PRV vaccine virus(gE-),a multiplex nano-PCR method for simultaneous detection of ASFV,PRV wild virus and PRV vaccine virus were established by using the B646L gene of ASFV and the gE and gD gene of PRV as the detection objects.The method's specificity and sensitivity were studied and the results showed that it could specifically amplify the target genes of ASFV,PRV wild virus and PRV vaccine strain,but the re-sults were negative when detecting Seneca virus,porcine haemagglutinating encephalomyelitis virus,porcine vesicular stomatitis virus and porcine circovirus type 2.The minimum detection limits of B646L gene,gD gene and gE gene were 4,7 and 8 copies/μL,respectively.In addition,the effectiveness of the multiplex nano-PCR detection method was compared with commonly used commercial kits for these viruses,and the results showed that the coincidence rate was 100%.In summary,the multiplex nano-PCR method established in this experiment has high specificity and sensitivity and could simultaneously detect ASFV,wild-type and vaccine PRV,which could be used for differential diagnosis and disease prevention and control of major diseases inpigs.