鉴别猪流行性腹泻病毒野毒株与疫苗株双重RT-PCR检测方法的建立

Development of duplex RT-PCR method for the differentiation of virulent and vaccine strains of porcine epidemic diarrhea virus

为建立鉴别猪流行性腹泻病毒野毒株与疫苗株的检测方法,本研究根据GenBank中登录的猪流行性腹泻病毒(PEDV)M、ORF3基因序列,设计合成了2对特异性引物,通过PCR扩增条件的优化,建立了鉴别PEDV野毒株和疫苗株的双重RT-PCR检测方法。特异性试验显示,该检测方法对猪圆环病毒2型、猪伪狂犬病毒、猪细小病毒、Salmonella及E.coli等结果均为阴性;敏感性试验显示,所建立的双重PCR检测方法对M基因和ORF3基因的最低有效检测量分别为44.5拷贝/μL和481拷贝/μL。本研究建立的双重RT-PCR检测方法可实现对PEDV野毒和疫苗毒的快速检测,适合于现地猪流行性腹泻的鉴别诊断。

To establish a rapid assay for virulent and vaccine strains differential of porcine epidemic diarrhea virus （PEDV）, a duplex RT-PCR method was developed with two pairs of primers according to the M and ORF3 genes of PEDV available in GenBank. Under the optimized conditions, two specific DNA fragments of 419 bp for M gene and 247 bp for ORF3 gene were simultaneously amplified from PEDV virulent strain; while only a 419 bp fragment for M gene was amplified from PEDV vaccine strain. Meanwhile, no amplifications were found from porcine circovirus type 2, pseudorabies virus, porcine parvovirus, Salmonella or E.coli DNA. In addition, the duplex PCR method was capable of detecting up to the template of 44.5 copies/ixL for M gene and 481 copies/μL for ORF3 gene. The established duplex PCR method is suitable for differentiation of wild-type and vaccine PEDV strains in clinical samples, which provides effective technical support for the differential diagnosis of PEDV infection.