猪传染性胃肠炎病毒SYBR Green I模式实时定量RT-PCR检测方法的建立及初步应用

登录GenBank下载猪传染性胃肠炎病毒(TGEV)的N基因序列,依据保守区设计一对特异性引物,采用SYBR GreenⅠ随机结合渗入法,建立检测猪传染性胃肠炎病毒的实时定量RT-PCR检测方法,成功构建了检测TGEV的标准DNA模板,循环阈值(Ct)与标准DNA模板在1.0×10-1.0×107拷贝/μl浓度范围内呈良好的线性关系,相关系数为0.9985。该方法用于检测TGEV具有很高的特异性,其敏感性与常规PCR相比可以提高100倍,可以用于猪传染性胃肠炎病毒的快速检测。用建立的检测方法对82份临床样品进行了检测,和普通PCR检测结果100%相符。

Development of SYBR Green I Real-time RT-PCR Method for Transmissible Gastroenteritis Virus and Its Application

One pair of primers specific based on the conservative region of TGEV N gene sequences in Gen-Bank was designed.With SYBR Green Ⅰ random conjugated incorporation method,a real-time quantitative PCR method was established to detect TGEV standard DNA templates.A good linearity relationship was showed between cycle threshold and standard DNA templates at 1.0×10-1.0×107copies/ul,and the correlation coefficient was 0.9985.The method was of high specificity and could be used to detect TGEV rapidly,which has 100 times higher than PCR in sensitivity.The results showed 100 percent agreement for the detection of 82 clinical samples with the established method and the common PCR method.