PCV-2、PPV和PRV多重PCR检测方法的建立及初步应用

Establishment and initial application of multiplex PCR assay for detecting PCV-2,PPV and PRV infection

【目的】建立可用于临床同时检测PCV-2、PPV和PRV 3种DNA病毒感染的多重PCR方法。【方法】根据GenBank中收录的PCV-2、PPV和PRV的基因组序列,选择3种病毒的特异性保守区设计3对引物,通过优化多重PCR的反应条件,建立了能够同时检测3种病毒混合感染的多重PCR方法,对该方法的特异性、敏感性、稳定性进行检测,并将其用于临床病料的检测。【结果】多重PCR方法中,当引物浓度均为1.0μmol/L,退火温度为57.2℃时,各目的片段均可较好地扩增。特异性试验结果表明,建立的多重PCR方法可从PCV-2、PPV、PRV病毒DNA中分别扩增出长度为353,265和198bp的目的片段,从3种病毒DNA的混合物中也可扩增出上述目的片段,而其他对照组的扩增结果均呈阴性。敏感性试验结果表明,建立的多重PCR方法对PCV-2、PPV和PRV的最低检测量分别为26.88,25.34和24.9pg/μL。在不同时间对每份样品重复检测3次,结果一致,表明该方法具有良好的可重复性。临床应用结果表明,39份疑似病料中,PCV-2、PPV和PRV的阳性率分别为53.84%,17.95%和5.13%,PCV-2与PPV混合感染的阳性率为15.38%,PCV-2、PPV和PRV混合感染的阳性率为2.57%。【结论】建立的多重PCR方法敏感性高、特异性强、重复性好,可以有效检测PCV-2、PPV和PRV的混合感染。

【Objective】 A multiplex polymerase chain reaction assay was developed for the clinical detection of porcine circovirus type 2,porcine pseudorabies virus and porcine parvovirus infection of swine.【Method】 According to the highly conserved genome sequence of PCV-2,PPV and PRV,three pairs of primers targeting PCV-2 ORF2,PPV NS1 and PRV gB were synthetized.The multiplex PCR reaction condition was optimized and the multiplex PCR for simultaneous detecting PCV-2,PPV and PRV was established.The specificity,sensitivity and repeatability of the multiplex PCR were detected and the multiplex PCR method was applied to clinical sample detection.【Result】 All target fragments were well amplified,when the primers concentrations were 1.0 μmol/L and the annealing temperature was 57.2 ℃;The specificity test showed that a fragment of 353,265 and 198 bp was amplified from the genomic DNA of PCV-2,PPV and PRV respectively.Three fragments were amplified from the mixed DNA sample of PCV-2,PPV and PRV simultaneously,and no amplification was achieved from other negative control groups.The sensitivity test showed that the multiplex PCR of PCV-2,PPV and PRV DNA could be detected as 26.88,25.34 and 24.9 pg/μL;The results of 3 replication test for each sample at different time were consistent indicating that multiplex PCR had good repeatability.The initial application test showed that the 39 clinical samples were subjected to multiplex PCR.Among these samples,PCV-2 positive percentage was 53.84%,PPV positive percentage 17.95% and PRV positive percentage 5.13%,co-infection of PCV-2 and PPV percentage 15.38%,co-infection of PCV-2,PPV and PRV percentage 2.57%.【Conclusion】 These results indicated that multiplex PCR had specificity,sensitivity,and repetitive characteristics and multiplex PCR had potential values for detecting of PCV-2,PPV and PRV co-infection in pigs.