PCV3和PCV2双重PCR鉴别诊断方法的建立及应用

Establishment and application of the multiplex PCR to detect and discriminate PCV3 and PCV2

本研究根据GenBank中报道的PCV2和PCV3毒株全基因组序列,设计两对特异性引物,建立了一种多重PCR鉴别诊断方法,两种病毒的预期扩增片段分别大小分别为1 007 bp和505 bp。通过对扩增条件的优化,获得最佳的反应体系为25μL体系,最佳退火温度为56℃。该鉴别方法可以同时扩增PCV3和PCV2的特异性片段,而猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)、猪繁殖障碍与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)、猪乙型脑炎病毒(JEV)、轮状病毒(RV)、猪流行性腹泻病毒(PEDV)和猪德尔塔冠状病毒的基因组及阴性对照均未扩增出任何条带。该方法对PCV3和PCV2的最低检出量均为10 ng/μL。对采自湖北省内规模化猪场的260份淋巴结样品检测。结果显示,PCV3阳性率为16.9%(44/260),PCV2阳性率为46.5%(121/260),PCV3和PCV2混合感染率为4.6%(12/260),双重PCR检测与测序分析的符合率为100%。结果表明,本研究建立了一种简便、灵敏、稳定可靠且特异性强的双重PCR鉴别诊断方法,可为临床样品中PCV3和PCV2的快速鉴别诊断及流行病学研究提供技术支持。

In this study, two pairs of specific primers were designed based on the published sequence of the complete genome of PCV3 and PCV2 in GenBank. And the duplex PCR was successfully established by optimizing the duplex PCR amplification conditions. The specific PCR products of1 007 bp for PCV3 and 505 bp for PCV2 were simultaneously amplified from the DNAs of PCV3 and PCV2 by the duplex PCR, but no PCR products were amplified from classical swine fever virus(CSFV), pseudorabies virus(PRV), porcine reproductive and respiratory syndrome virus(PRRSV), porcine parvovirus(PPV), Japanese encephalitis virus(JEV), rotavirus(RV), porcine epidemic diarrhea virus(PEDV), porcine deltacorona virus(PDCoV), and negative control. The duplex PCR method was capable of detecting the template DNA of 10 ng/μL for both PCV3 and PCV2. 260 lymph node samples from Hubei were detected by the duplex PCR assay. And the positive samples were detected by sequencing method. The results showed that PCV3 and PCV2 infection was rather common, with 16.9% positive for PCV3, 46.5% positive for PCV2, and 4.6% positive for PCV3 and PCV2 co-infection. The coincidence between duplex PCR assay and sequencing method was 100%. The results indicated that the duplex PCR was characterized by convenience, high sensitivity, stability and specificity, which can provide effective support for differentiating PCV3 and PCV2 infection in clinical samples and the epidemiologic study for PCV3 and PCV2.