猪繁殖与呼吸综合征病毒2型TaqMan MGB探针荧光定量PCR方法的建立

Establishment of a TaqMan MGB probe fluorescence quantitative PCR method for porcine reproductive and respiratory syndrome virus type 2

为了准确、特异、高效地检测猪繁殖与呼吸综合征病毒2型(PRRSV2),本研究根据GenBank上登录的PRRSV2不同谱系流行毒株的ORF6基因序列,选取保守区域设计合成了1对特异性检测引物和1条MGB探针,经优化退火温度、引物和探针比例等反应条件及敏感性、特异性、重复性试验,建立了检测PRRSV2的TaqMan MGB荧光定量PCR方法。结果:以猪繁殖与呼吸综合征病毒1型(PRRSV1)、伪狂犬病病毒(PRV)、猪瘟病毒(CSFV)、猪圆环病毒2型(PCV2)、猪圆环病毒3型(PCV3)、猪流行性腹泻病毒(PEDV)及猪细小病毒(PPV)等阳性核酸为模板,均无扩增;对标准质粒的最低检测下限为1×10^(1)copies/μL,建立的标准曲线呈现良好的线性关系,相关系数R2为0.994;稳定性和重复性好,批内和批间变异系数均小于1.78%。应用该方法对保留的30份临床阳性样品进行检测,结果阳性100%,Ct值在22~36间。综上,本研究建立的TaqMan MGB荧光定量PCR方法可用于临床和实验室中PRRSV2的快速检测,为猪繁殖与呼吸综合征的防控提供了技术支持。

In order to establish an accurate,specific,efficient and rapid fluorescence quantitative PCR method for the detection of porcine reproductive and respiratory syndrome virus type 2(PRRSV2),a pair of specific primers and a MGB probe were designed and synthesized according to the ORF6 gene of different lineage PRRSV2 strains registered in GenBank.Then,a TaqMan MGB fluorescence quantitative PCR method for the detection of PRRSV2 was established by optimizing the conditions and systems of reaction,primer and probe ratio,and by conducting sensitivity,specificity and reproducibility experiments.The results showed that the method was highly specific and did not amplify the nucleic acid of PRRSV1,classical swine fever virus(CSFV),porcine pseudorabies virus(PRV),porcine circovirus type 2(PCV2),porcine circovirus type 3(PCV3),porcine epidemic diarrhea virus(PEDV),porcine parvovirus(PPV)and other pathogens.The minimum detection limit of the standard plasmid was 1×10^(1)copies/μL.The standard curve showed a good linear relation,and the correlation coeffi⁃cient was(R2=0.994),with good stability and reproducibility.The intra-and inter-assay coefficients of variation were less than 1.78%.30 clinical positive samples were also positive using this method with Ct values between 22 and 36.In summary,the PRRSV2 TaqMan MGB flu⁃orescence quantitative PCR method established in this study could be used for rapid detection of PRRSV2 in clinical practice and laboratory trial,providing technical support for PRRS control.