猪流行性腹泻病毒和猪博卡病毒3/4/5型双重PCR检测方法的建立

Development of duplex PCR for detection of porcine epidemic diarrhea virus and porcine bocavirus genetypes 3/4/5

为建立快速检测猪流行性腹泻病毒(PEDV)和猪博卡病毒(PBoV)3/4/5型的双重PCR方法,本研究根据Gen Bank中登录的PEDV ORF1基因序列和PBoV不同基因型VP1序列,设计2对特异性引物,通过对PCR扩增条件的优化,建立了能够同时检测PEDV和PBoV3/4/5型双重PCR方法,特异性检测结果显示该方法对猪细小病毒、猪圆环病毒2型、猪伪狂犬病毒、大肠杆菌、沙门氏菌、猪链球菌核酸扩增均为阴性;敏感性检测结果显示,对PEDV和PBoV3/4/5型重组质粒标准品的最低检出量分别为837拷贝/μL和1 000拷贝/μL;临床样品的检测结果显示,所建立的双重PCR方法可同时有效地检测出PEDV和PBoV3/4/5型混合感染及单独感染。本研究建立的双重PCR方法具有良好的特异性、敏感性、重复性,为快速、高效检测PEDV和PBoV提供了技术帮助。

To develop a rapid duplex PCR assay for simultaneous detection of porcine epidemic diarrhea virus （PEDV） and porcine bocavirus （PBoV） 3/4/5 genotypes, two pairs of primers were designed and synthesized according to the published sequences of the ORF1 gene of PEDV and the VP1 gent of PBoV 3/4/5 genotypes in GenBank, and a duplex PCR protocol for detection of PEDV and PBoV3/4/5 was developed by optimizing conditions of the PCR reaction. The specific fragments of 163 bp for PEDV and 135 bp for PBoV3/4/5 were simultaneously amplified in the duplex PCR, but no PCR products were amplified for porcine parvovirus, porcine circovirus type 2, pseudorabies virus, Escherichia coli, Salmonella, S.suis. The detection limit was 837 copies/txL of PEDV and 1 000 copies/ixL of PBoV3/4/5, respectively. Clinical detection of PEDV and PBoV3/4/5 by duplex PCR indicated that the duplex PCR had high specificity and sensitivity, which provides effective support for detecting the clinical samples with PEDV and PBoV or co-infection.