新现猪圆环病毒3型及圆环病毒2型双重PCR检测方法的建立与应用

Establishment and Application of Double PCR Assays for Detection of New-emerged Porcine Circovirus Type 3(PCV-3)and Porcine Circovirus Type 2(PCV-2)

根据GenBank数据库中的新现猪圆环病毒3型(PCV-3)和猪圆环病毒2型(PCV-2)的OFR2基因设计了2对扩增PCV-3和PCV-2的特异性引物,通过优化各反应条件,建立同时检测PCV-3和PCV-2的双重PCR方法。敏感性和特异性结果显示,该方法对PCV-3和PCV-2的最低核酸检测量均为1.0×103拷贝/μL,而对猪伪狂犬病毒(PRV)、猪delta冠状病毒(PDCoV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪繁殖与呼吸综合征病毒(PRRSV)及猪瘟病毒(CSFV)的扩增结果均为阴性。应用所建立的双重PCR检测了临床上256份样品,结果表明,PCV-3和PCV-2感染的阳性率分别为0.78%(2/256)和86.3%(221/256),仅有1份样品为PCV-3和PCV-2混合感染,混合感染阳性率为0.39%(2/256)。试验结果表明,本研究建立的PCR方法敏感、特异,适于临床样品检测,为猪群中PCV-3和PCV-2的临床快速诊断和流行病学调查提供了一种工具。

According to the published ORF2 gene sequences of new emerged porcine circovirus 3（PCV-3）and porcine circovirus 2（PCV-） in GenBank,two pairs of primers specific to PCV-3 and PCV-2 were designed and synthesized.By optimizing the reaction conditions,duplex polymerase chain reaction（PCR）.based assay was developed and evaluated for its ability to simultaneously detect co.viral infections of swine.The detection limit of the established assay was 1.0 x10^3 copies/μL for both PCV-3 and PCV-2.When common swine viral pathogens,including pseudorabies virus（PRV）,porcine deltacoronavirus（PDCoV）,porcine epidemic diarrhea virus（PEDV）,porcine transmissible gastroenteritis virus（TGEV）,porcine reproductive and respiratory syndrome virus（PRRSV） and classical swine fever virus（CSFV）,served as the template for the evaluation of the specificity of the assay,no cross amplification was observed.Based on the clinical investigation on 256 samples,the positive rates of PCV-3 and PCV-2 were 0.78%（2/256） and 86.3%（221/256）,respectively.Only one positive sample was co.infected with PCV-3 and PCV-2.This study showed that the duplex PCR established in this study was sensitive and specific,and would be a useful tool for clinical diagnosis and epidemiologic survey of PCV-3 and PCV-2 circulating in swine.