猪圆环病毒2型猪伪狂犬病病毒和猪细小病毒三重PCR检测方法的建立

Development of a triplex PCR assay for detection of porcine circovirus type 2,pseudorabies virus and porcine parvovirus

针对猪圆环病毒2型(PCV2)ORF2、猪伪狂犬病病毒(PRV)gD和猪细小病毒(PPV)VP2基因保守区域,设计了特异性引物,在分别建立单一病毒基因PCR检测方法的基础上,通过对3组引物的比例和浓度、dNTPs和Mg2+浓度、退火温度等条件的优化,建立了针对上述3种病毒的三重PCR检测方法。敏感性试验表明,应用该方法最低可检测到4×10-4ng/μL的PRV双链DNA模板和2×10-5ng/μL的PCV2和PPV单链DNA模板。对21份自然感染病猪样品的检测结果表明,该三重PCR检测结果与单一PCR检测的结果完全符合。试验结果显示,建立的三重PCR方法可用于3种DNA病毒的同时检测,具有快速、准确的特点,有临床应用前景。

A triplex PCR assay was developed and evaluated for the simultaneous detection of infections with porcine circovirus type 2（PCV2）, pseudorabies virus（PRV） and porcine parvovirus（PPV）. Specific primers for each of the three viruses were designed and used. The lowest detection limit of the triplex PCR was 4× 10^-4 ng/μL approximately for double-strained DNA template of PRV and 2 × 10^-5 ng/μL for single-strained DNA template of PCV2 and PPV, when a composite of the three viruses was tested as a single sample. This assay was also effective for detecting one or two of the three viruses in various combinations in 21 tissue specimens collected from diseased pigs and aborted fetuses. Results suggested that the established triplex PCR assay had potential for clinic application.