猪流行性腹泻病毒TaqMan荧光定量PCR方法的建立与应用

Development and application of TaqMan-based real-time PCR assay for detection of porcine epidemic diarrhea virus

为定量检测猪流行性腹泻病毒（Porcine epidemic diarrhea virus,PEDV）,建立了检测PEDV的TaqMan荧光定量PCR方法.根据PEDV基因组中保守的N基因序列设计引物和探针,以梯度稀释的含有N基因的重组质粒作为标准品,进行定量PCR反应.结果表明,该试验建立的荧光定量PCR方法在1μl 10^1 ～ 10^9拷贝之间具有良好的线性关系,相关系数达到0.99以上,扩增效率在90％与100％之间.该方法的检测灵敏度为10拷贝,与常规RT-PCR方法相比,该方法敏感性更高,而且该检测方法特异性较好,与猪的其他病毒核酸均无交叉反应,批内和批间的变异系数均低于3％,表明该方法的重复性较好.对华东地区采集的130份临床样品进行检测,结果显示,PEDV的阳性率为63.1％.

The TaqMan-based real-time PCR assay was developed for rapid and sensitive detection of porcine epi-demic diarrhea virus （PEDV）. The primers and probe were designed targeting the PEDV N gene. The recombinant plasmidcontaining the N gene was constructed to establish the standard curve. The results showed that the assay had a dynamicrange of detection between 101 -109 copies, with the correlation coefficient of more than 0. 99, and the amplification effi-ciency between 90% and 100%. The real-time PCR was sensitive for PEDV with a detection limit of 10 copies, which wasbetter than traditional RT-PCR. The real-time PCR was specific for PEDV; it could not amplify any other porcine virus.And the PCR presented good repeatability, with thecoefficiencts of inter-and intro-variation less than 3%. Atotal of 130 clinical samples collected from eastern Chinawere tested, and the positive rate of PEDV was 63. 1%. Itwas concluded that TaqMan-based PCR for detection and quantification of PEDV was highly sensitive and specific, which would provide a reliable protocol for the epidemiologicalsurvey.