反转录巢式PCR方法检测SARS冠状病毒  

Gene sequence analysis of SARS-associated coronavirus by nested RT-PCR

作　　者：王艳[1] 马文丽[1] 宋艳斌[1] 肖维威[1] 张宝[1] 黄海[1] 王洪敏[1] 马晓冬[1] 郑文岭[2] 

机构地区：[1]第一军医大学基因工程研究所,广东广州510515 [2]广州军区广州总医院医学实验科,广东广州510010

出　　处：《第一军医大学学报》2003年第5期421-423,共3页Journal of First Military Medical University

基　　金：全军十五重大攻关项目资助~~

摘　　要：目的探索SARS冠状病毒早期诊断的有效手段。方法对临床病例样品采用反转录巢式PCR方法鉴定SARS冠状病毒。提取样品RNA,用巢式PCR外侧下游引物进行反转录,以反转录产物为模板,进行巢式第一次、第二次PCR。PCR产物克隆到pMD18-T载体后进行测序鉴定。结果被检样品扩增出特异片段,经测序验证确实来自SARS相关冠状病毒。Blast比较结果与SARS冠状病毒多伦多株相应片段只有一个碱基的差异。结论反转录巢式PCR方法验证在非典型肺炎患者标本中存在SARS相关冠状病毒的序列,证实该法不失为一种检测SARS冠状病毒的快速、简便、易行的方法。Objective To explore an effective means for th e detection of Severe Acute Respiratory Syndrome (SARS)-associated coronav irus. Method The RNAs of the virus contained in the sputum samples from establis hed SARS patients were extracted and reversely transcripted, followed by nested PCR using the reversely transcripted cDNA as the template. The PCR products we re cloned then into the pMD18-T vectors, followed by sequence analysis. Result S pecific fragments were amplified from the sputum samples of SARS patients, whic h were confirmed by DNA cloning and sequencing to belong to SARS-associated cor onavirus. The Result of Blast shows only the difference in one nucleic acid from the TOR2 strain of SARS-associated coronavirus. Conclusion Sequence analysis h as confirmed the existence of SARS-associated coronavirus in the sputum samples of SARS patients, and nested RT-PCR is a quick, easy, and convenient way for th e detection of the virus.

关 键 词：SARS冠状病毒 巢式反转录PCR 早期诊断 

分 类 号：R346[医药卫生—基础医学]