一步法双重RT-PCR检测SARS冠状病毒技术研究

One-step Duplex RT-PCR Assay for Detection SARS Associated Coronavirus

参照WHO公布的PCR检测SARS相关冠状病毒的引物序列,我们合成了4对引物,建立了一步法RT-PCR检测SARS相关冠状病毒的方法。利用该方法,对武汉市非典型肺炎疑似病例喉漱液进行了SARS相关冠状病毒检测,其中w20用4对引物检测均在相应的位置出现了DNA条带,W1只有BNIin-s/as引物出现预期产物。同时我们将样品W20的RT-PCR扩增产物进行了序列测定,结果显示该片段序列与其他地区和国家分离的SARS冠状病毒基因高度同源性,这进一步说明RT-PCR扩增的产物是SARS冠状病毒基因片段。在此基础上我们又建立了四种SARS冠状病毒一步法双重RT-PCR检测方法,在一个RT-PCR反应中用两套引物同时对SARS冠状病毒基因两个不同的片段进行检测,检测结果具有更高的特异性。

According to PCR primers for SARS developed by WHO network laboratories we synthesize 4 primer pairs: BNIin-s/as、BNIout-s/as、SARl-s/as、COR1/COR2 and establish assays of one-step RT-PCR for detection of SARS associated coronavirus. Specific DNA fragments as predicted were produced from specimen W20 by the assays using all 4 primer pairs, while one specific DNA fragment was produced from specimen W1 using primer pair BNIin-s/as. The PCR products from W20 were sequenced and the sequence showed high identity to that of all SARS associated coronavirus in GenBank, which showed that PCR products amplified from W1 and W20 by our assays was specific for SARS associated coronavirus. Based on the one-step RT-PCR we developed four one-step duplex RT-PCR for amplifying two fragment in one reaction by combination of 4 primer pairs above. And the assays of one-step duplex RT-PCR have higher specificity and were reliable for detection of SARS associated coronavirus.