猪圆环病毒3型和4型TaqMan双重实时荧光定量PCR检测方法的建立

Establishment of duplex Taqman real-time PCR method for detection of porcine circovirus type 3 and 4

为了建立一种快速检测猪圆环病毒3型(PCV3)和4型(PCV4)的TaqMan双重实时荧光定量PCR检测方法,根据Gen Bank已登录的PCV3和PCV4基因保守序列设计特异性引物和TaqMan探针,经优化各反应条件,构建标准曲线,分析其敏感性、特异性和重复性,并利用建立的荧光定量PCR方法对采自安徽省的临床样品进行检测,与普通PCR方法进行一致性评价。结果显示,PCV3的标准曲线为Y=–3.534 6 logX+41.11(R^(2)=1.00),PCV4的标准曲线为Y=–3.382 5 logX+40.67(R^(2)=1.00)。PCV3和PCV4的检测限分别为49.5和27.1 copies·μL^(-1)。对猪圆环病毒1型(PCV1)和2型(PCV2)、猪流行性腹泻病毒(PEDV)、猪瘟病毒(CSFV)、伪狂犬病毒(PRV)及猪繁殖与呼吸综合征病毒(PRRSV)无特异性扩增,组内和组间变异系数均<1%。临床样品检测结果显示,PCV3阳性检出率为40.8%,PCV4阳性检出率为20.4%,混合感染率为19.0%。建立了灵敏、特异,可同时快速、准确鉴别PCV3和PCV4的TaqMan双重实时荧光定量PCR检测方法,为PCV3和PCV4的检测和监测提供技术支撑。

To establish a duplex TaqMan real-time PCR method for the rapid detection of porcine circovirus type 3(PCV3)and type 4(PCV4),the specific primers and TaqMan probes were designed based on the conserved sequences of PCV3 and PCV4 genes registered in GenBank.The reaction conditions were optimized,and the con-struction of standard curves was carried out.The sensitivity,specificity and reproducibility were analyzed,and an established fluorescence quantitative PCR method was used to test clinical samples collected from Anhui Province to evaluate its consistency with the general PCR method.The results showed that the standard curve for PCV3 was Y=-3.5346 logX+41.11(R^(2)=1.00)and for PCV4 was Y=-3.3825 logX+40.67(R^(2)=1.00).The lower limits of detection for PCV3 and PCV4 were 49.5 and 27.1 copies·μL^(-1),respectively.There was no specific amplifica-tion for porcine circovirus type 1(PCV1)and type 2(PCV2),porcine epidemic diarrhea virus(PEDV),classic swine fever virus(CSFV),pseudorabies virus(PRV),and porcine reproductive and respiratory syndrome virus(PRRSV),with intra-and inter-batch coefficients of variation(CV)less than 1%.The results of the clinical sam-ples showed that the detection rate was 40.8%for PCV3,20.4%for PCV4 and 19.0%for co-infection.As results,a sensitive and specific duplex TaqMan real-time fluorescent quantitative PCR method was established,which can quickly and accurately identify PCV3 and PCV4 simultaneously and provide some technical supports for the de-tection and monitoring of PCV3 and PCV4.