中东呼吸综合征冠状病毒半巢式PCR检测方法的建立
发布时间：2019-05-05 12:37
【摘要】：目的建立特异、灵敏的半巢式PCR方法以期用于检测中东呼吸综合征冠状病毒(MERS-CoV)。方法根据MERS-CoV N蛋白基因设计并合成特异性内侧、外侧引物。通过条件优化,建立MERS-CoV半巢式PCR检测体系,扩增产物测序后与目的序列进行同源比对。以10倍系列稀释重组质粒为标准品,与普通PCR反应进行比较,检测半巢式PCR方法的灵敏度。以其他6种呼吸道病原体及冠状病毒基因为对照,检测半巢式PCR方法的特异性。结果使用建立的半巢式PCR方法扩增的特异片段与GenBank中发表的MERS-CoV基因序列同源性为100%;半巢式PCR检测方法的灵敏度为1.17×101拷贝,比普通一轮PCR扩增的灵敏度(1.17×105拷贝)提高了104倍;用该方法检测MERS-CoV基因为阳性,其他6种呼吸道病原体及冠状病毒基因检测均呈阴性。结论建立的MERS-CoV半巢式PCR方法具有良好的准确性、灵敏性和特异性,为MERS-CoV感染的诊断提供了一种新的、可靠的检测手段。
[Abstract]:Objective to establish a specific and sensitive semi-nested PCR method for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV). Methods specific medial and lateral primers were designed and synthesized according to MERS-CoV N protein gene. The MERS-CoV semi-nested PCR detection system was established by optimizing the conditions. The amplified products were sequenced and compared with the target sequence. The sensitivity of semi-nested PCR method was compared with that of ordinary PCR using 10-fold diluted recombinant plasmid as standard. Six other respiratory pathogens and coronavirus genes were used as controls to detect the specificity of the semi-nested PCR method. Results the specific fragment amplified by the semi-nested PCR method shared 100% homology with the published MERS-CoV gene sequence in GenBank. The sensitivity of semi-nested PCR was 1.17 脳 101copies, which was 104 times higher than that of conventional PCR amplification (1.17 脳 105copies). The other 6 respiratory pathogens and coronavirus genes were negative for the detection of MERS-CoV gene by this method. Conclusion the established MERS-CoV semi-nested PCR method has good accuracy, sensitivity and specificity, which provides a new and reliable method for the diagnosis of MERS-CoV infection.
【作者单位】： 军事医学科学院军事兽医研究所;吉林省人兽共患病预防与控制重点实验室;江苏省动物重要疫病与人兽共患病防控协同创新中心;中国农业科学院生物技术研究所;
【基金】：国家科技支撑计划项目(No.2013BAD12B04) 中国博士后科学基金项目(No.2013M541089)
【分类号】：R440;R511
，
本文编号：2469569
[Abstract]:Objective to establish a specific and sensitive semi-nested PCR method for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV). Methods specific medial and lateral primers were designed and synthesized according to MERS-CoV N protein gene. The MERS-CoV semi-nested PCR detection system was established by optimizing the conditions. The amplified products were sequenced and compared with the target sequence. The sensitivity of semi-nested PCR method was compared with that of ordinary PCR using 10-fold diluted recombinant plasmid as standard. Six other respiratory pathogens and coronavirus genes were used as controls to detect the specificity of the semi-nested PCR method. Results the specific fragment amplified by the semi-nested PCR method shared 100% homology with the published MERS-CoV gene sequence in GenBank. The sensitivity of semi-nested PCR was 1.17 脳 101copies, which was 104 times higher than that of conventional PCR amplification (1.17 脳 105copies). The other 6 respiratory pathogens and coronavirus genes were negative for the detection of MERS-CoV gene by this method. Conclusion the established MERS-CoV semi-nested PCR method has good accuracy, sensitivity and specificity, which provides a new and reliable method for the diagnosis of MERS-CoV infection.
【作者单位】： 军事医学科学院军事兽医研究所;吉林省人兽共患病预防与控制重点实验室;江苏省动物重要疫病与人兽共患病防控协同创新中心;中国农业科学院生物技术研究所;
【基金】：国家科技支撑计划项目(No.2013BAD12B04) 中国博士后科学基金项目(No.2013M541089)
【分类号】：R440;R511
，
本文编号：2469569
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