猪伪狂犬病病毒、猪圆环病毒2型和3型三重PCR检测方法的建立

Development of a multiplex PCR for detection of PRV,PCV2 and PCV3

为了建立能够同时检测猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)和猪圆环病毒3型(PCV3)单一或混合感染的PCR检测方法,参考GenBank中PRV gE基因、PCV2和PCV3全基因序列,针对PRV gE、PCV2 Rep和PCV3 Rep基因的保守区域,利用Primer 5.0设计3对特异性引物,随后对其扩增条件进行优化,建立能够同时检测PRV、PCV2和PCV3的三重PCR方法。该方法能同时扩增PRV的429 bp、PCV2的273 bp和PCV3的344 bp特异性片段,而对常见的其他猪病原扩增均为阴性。PRV、PCV2和PCV3的最低检出量分别为1 250.0、693.0和91.2拷贝/μL。运用三重PCR和单项PCR检测疑似患有母猪繁殖障碍或呼吸系统疾病的74份临床病料,发现PRV、PCV2和PCV3的阳性率分别为29.73%(22/74)、71.62%(53/74)和58.11%(43/74),且三重PCR与单项PCR检测结果的符合率为100%。本试验建立了能够同时检测PRV、PCV2和PCV3的三重PCR检测方法,且该方法具有良好的特异性、灵敏性和准确性,为PRV、PCV2和PCV3的临床诊断和监测提供技术支持。

To develop a rapid multiplex PCR assay for simultaneous detection of porcine pseudorabies virus(PRV),porcine circovirus type 2(PCV2) and porcine circovirus type 3(PCV3).Three pairs of primers were designed using Primer 5.0 software according to the conserved regions of the gE gene of PRV,Rep gene of PCV2 and PCV3 published in GenBank, then a multiplex PCR method was established to detect PRV,PCV2 and PCV3 simultaneously after the amplification conditions were optimized.This method could simultaneously amplify specific fragments of 429 bp for PRV,273 bp for PCV2 and 344 bp for PCV3,while the amplification results were negative for the pathogens of other common pig diseases.The minimum detectable values were 1 250.0,693.0 and 91.2 copies/μL for PRV,PCV2 and PCV3,respectively.Seventy-four clinical samples of pigs affected with reproductive failure or respiratory disorders were tested to evaluate the multiplex PCR and single PCR,and the positive rates of PRV,PCV2 and PCV3 were 29.73%(22/74),71.62%(53/74) and 58.11%(43/74),respectively.In addition, the coincidence rate between multiplex PCR and single PCR was 100%.The results indicate that this multiplex PCR assay has excellent specificity and sensitivity, and can provide technical support for the detection of PRV,PCV2 and PCV3 in the clinical samples.