一步法RT-PCR检测SARS冠状病毒技术研究

One-step RT-PCR for detection of SARS associated coronavirus

〔目的〕建立一步法RT -PCR方法 ,对非典型肺炎疑似病例进行SARS相关冠状病毒检测。〔方法〕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 ,利用RT -PCR技术建立一步法检测SARS相关冠状病毒的方法。〔结果〕利用该方法对武汉市非典型肺炎疑似病例进行SARS相关冠状病毒检测 ,其中样品W2 0用 4对引物检测均在相应的位置出现了DNA条带 ,样品W 1只有BNIin -s/as引物出现预期产物。同时我们将W2 0的BNIin -s/as引物扩增的 10 9bp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的SARS冠状病毒的同源性极高 ,均在 99.5 %以上。〔结论〕一步法RT -PCR扩增产物是SARS冠状病毒基因片段 ,我们建立的一步法RT -PCR是一种可靠的检测方法。

〔objective〕 To establish assays of one-step RT-PCR for detection of SARS associated coronavirus;〔Method〕 According to PCR primers for SARS developed by WHO network laboratories we synthesize 4 primer pairs: BNIin-s/ as?BNIout-s/as?SAR1-s/ as?COR1/COR2 and establish assays of one-step RT-PCR for detection of SARS associated coronavirus by RT-PCR technology.〔Results〕 20 specimens from SARS probable cases in Wuhan were test by the one-step RT-PCR assay, 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 primer pair BNIin-s/ as PCR products from W1 and W20 were sequenced and the sequence showed high identity to that of all SARS associated coronavirus in GenBank; 〔Conclusions〕The results showed that PCR products from W1 and W20 by our assays was specific for SARS associated coronavirus and the assays of one-step RT-PCR for detection of SARS associated coronavirus established by us were reliable.