猪博卡病毒1/2型NS1基因与3/4/5型VP1基因的双重PCR检测方法的建立

Duplex PCR detection of porcine bocavirus genotype 1/2 by NS1 gene and porcine bocavirus genotype 3/4/5 by VP1 gene

为建立快速检测不同基因型猪博卡病毒(PBo V)的PCR方法,本研究参考Gen Bank中登录的5种基因型PBo V1、PBo V2、PBo V3、PBo V4及PBo V5,设计了2对引物,通过对扩增条件进行优化,建立了PBo V1/2和PBo V3/4/5的双重PCR检测方法。特异性试验结果表明,该方法可以同时扩增PBo V样品327 bp(PBo V1/2)和209 bp(PBo V3/4/5)的特异性片段,而对猪伪狂犬病毒、猪圆环病毒等其他病原核酸扩增均为阴性。敏感性试验显示,该PCR方法对PBo V1/2和PBo V3/4/5的最低检出量分别为100拷贝/μL和1 000拷贝/μL。重复性试验显示该双重PCR检测方法具有良好的可重复性。应用该方法和单项PCR对30份临床样品进行检测,结果显示,同时感染PBo V1/2和PBo V3/4/5的阳性率为10%,并且两种方法符合率为100%。本研究建立的方法适合对PBo V1/2和PBo V3/4/5的联合检测。

To establish the assay for porcine bocavirus （PBoV） detection, two pairs of primers were designed and synthesized according to the published sequences of different genotypes PBoV in the GenBank, a duplex PCR protocol was developed for detection of PBoV1/2 and PBoV3/4/5 by optimizing conditions of the PCR reaction. The specific fragments of 327 bp for PBoV1/2 and 209 bp for PBoV3/4/5 were simultaneously amplified in the duplex PCR, but no amplifications were found from DNA of other pathogens such as pseudorabies virus, porcine circovirus type 2. Moreover, the duplex PCR method was repeatability and capable of detecting the template DNA of 100 copies/l^L and 1,000 copies/vL for PBoV1/2 and PBoV3/4/5, respectively. A total of 30 clinical samples were detected by the established duplex PCR assay, and 3 samples were positive for PBoVI/2 and PBoV3/4/5 （10%）. The coincidence between single PCR assays and the duplex PCR is 100%, which provides an effective support for the combined detection of PBoV1/2 and PBoV3/4/5.