SARS冠状病毒实时荧光RT-PCR定量检测

Development of Real Time RT-PCR Assay for The Quantitation of SARS-Associated Coronavirus

为建立一种快速、准确、特异的SARS病毒RNA定量检测方法 ,根据复合探针荧光定量分析原理 ,对SARS病毒核酸进行实时荧光定量RT PCR检测 .借助计算机辅助 ,对SARS病毒基因靶序列以及检测引物和探针进行了优化筛选 ;利用体外转录SARS病毒RNA靶序列 ,对RT PCR反应的镁离子浓度参数进行了优化 ;比较Trizol法、磁珠法、Qiagen法、煮沸法等 4种方法提取RNA的检测效果 ,建立了样本处理方法 ;通过对构建的体外转录靶序列模型的检测 ,对本方法的灵敏度、特异性、定量线性关系、精确度等进行了评价 ,并通过对4 2例临床标本的检测对本方法的检测效果进行了评估 .通过克隆SARS病毒核酸靶序列并进行体外转录 ,获得了长度约 1 2kb的体外转录RNA靶序列 ;经优化 ,荧光RT PCR反应液中的Mg2 + 浓度以 4 0mmol/L为最好 ;RNA提取方法采用磁珠法效果较好 ;本方法的检测灵敏度最低可达 5个拷贝的体外转录RNA分子 ,特异性10 0 % ,Ct 值的CV值小于 5 % .对临床确诊的 4 2份SARS病人血清和漱口水标本的检测结果表明 ,该方法的检出率为 79% ,与荧光抗体检测法的符合率为 70 % .上述结果表明 ,该方法建立的荧光定量RT PCR技术能够快速准确、特异、敏感地对SARS病毒核酸进行定量分析 ,为临床SARS冠状病毒RNA的检测提供了新的 。

In order to develop an assay for rapid, specific, quantitative detection of SARS-associated coronavirus, the primers and quantitative probes were designed and applied to detect coronavirus, based on the principle of complex probes quantitative assay. Sensitivity, specificity, reproducibility and range of quantitation of this method were determined. The quantitative RT-PCR for coronavirus with complex probes and an easy to handle and high efficiency method for isolation RNA from sample were established. The detect limit is 5 copies RNA per reaction and no negative samples or other RNA/DNA were detected with this method. It allows for a high sample throughput. It shows a very good precision, the coefficient of variation of threshold cycle was less than 5%. 42 clinical SARS samples were detected with this quantitative RT-PCR, the rate of SARS samples can be detected was 79%. It can be concluded that the method established for quantitaion of SARS-CoV is highly sensitive,rapid and easy to handle and shows a very good reproducibility,it can be applied to clinical diagnosis.