猪流行性腹泻病毒恒温隔绝式荧光PCR检测方法的建立
发布时间：2018-09-19 17:24
【摘要】：本研究旨在建立检测猪流行性腹泻病毒(PEDV)的恒温隔绝式PCR(iiPCR)。采用靶向PEDV S基因的引物和探针,通过对引物、探针、Taq酶及反转录酶等进行优化,建立了检测PEDV的iiPCR,对其特异性、敏感性和稳定性进行评价,对15份PEDV阳性样本和7份阴性样本进行检测,比较与现有的荧光定量RT-PCR方法的符合率,并对115份仔猪腹泻样本进行检测。结果显示,iiPCR的最佳反应体系为:上、下游引物各3.5μL、探针0.25μL、Taq酶1μL、反转录酶1.25μL、Buffer A 25μL、模板2μL,补足DEPC水至50L,从样本处理到报告结果仅需1 h;该方法能检出样本中的PEDV,对TGEV、PPV、CSFV等无关病原不检出,检测下限为17.5 copiesμL,对5个稀释度的阳性标准品进行检测,3次重复结果一致,与现有荧光定量RT-PCR的总符合率为90.9%;本研究建立的iiPCR对仔猪腹泻样本中PEDV的检出率为22.6%(26/115)。综上可见,本研究成功建立了检测PEDV的iiPCR,为PED的诊断提供了更为便捷并可现场使用的可靠方法。
[Abstract]:The purpose of this study was to establish a isothermal isolated PCR (iiPCR). For detection of porcine epidemic diarrhea virus (PEDV). By using primers and probes targeting PEDV S gene, the specificity, sensitivity and stability of iiPCR, for detecting PEDV were evaluated by optimizing the primers, probes, Taq enzymes and reverse transcriptase. 15 PEDV positive samples and 7 negative samples were detected, and the coincidence rate was compared with the existing fluorescence quantitative RT-PCR method, and 115 piglets diarrhea samples were detected. The results showed that the best reaction system of iPCR was: Downstream primer 3.5 渭 L, probe 0.25 渭 L Taq 1 渭 L, reverse transcriptase 1.25 渭 L buffer A 25 渭 L, template 2 渭 L, complement DEPC water to 50 L, from sample processing to report result only 1 hour, this method can detect PEDV, in the sample not to TGEV,PPV,CSFV and other unrelated pathogens. The detection limit was 17.5 copies 渭 L, and the results of three repeats of five dilution positive standard samples were consistent with the total coincidence rate of fluorescence quantitative RT-PCR was 90.90.The detection rate of PEDV in diarrhea samples of piglets was 22.6% (26 / 115). It can be seen that the iiPCR, for detecting PEDV provides a more convenient and reliable method for the diagnosis of PED.
【作者单位】： 西南民族大学生命科学与技术学院;金瑞鸿捷(厦门)生物科技有限公司;
【基金】：十三五国家重点研发计划项目(2016YFD0500705)
【分类号】：S852.651
[Abstract]:The purpose of this study was to establish a isothermal isolated PCR (iiPCR). For detection of porcine epidemic diarrhea virus (PEDV). By using primers and probes targeting PEDV S gene, the specificity, sensitivity and stability of iiPCR, for detecting PEDV were evaluated by optimizing the primers, probes, Taq enzymes and reverse transcriptase. 15 PEDV positive samples and 7 negative samples were detected, and the coincidence rate was compared with the existing fluorescence quantitative RT-PCR method, and 115 piglets diarrhea samples were detected. The results showed that the best reaction system of iPCR was: Downstream primer 3.5 渭 L, probe 0.25 渭 L Taq 1 渭 L, reverse transcriptase 1.25 渭 L buffer A 25 渭 L, template 2 渭 L, complement DEPC water to 50 L, from sample processing to report result only 1 hour, this method can detect PEDV, in the sample not to TGEV,PPV,CSFV and other unrelated pathogens. The detection limit was 17.5 copies 渭 L, and the results of three repeats of five dilution positive standard samples were consistent with the total coincidence rate of fluorescence quantitative RT-PCR was 90.90.The detection rate of PEDV in diarrhea samples of piglets was 22.6% (26 / 115). It can be seen that the iiPCR, for detecting PEDV provides a more convenient and reliable method for the diagnosis of PED.
【作者单位】： 西南民族大学生命科学与技术学院;金瑞鸿捷(厦门)生物科技有限公司;
【基金】：十三五国家重点研发计划项目(2016YFD0500705)
【分类号】：S852.651
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