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

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1次、第2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 the effective assay of SARS - associated coronavirus as well as the diagnosis of SARS (severe acute respiratory syndrome). Methods RNAs of clinical samples from Beijing were extracted and reversed transcribed by out an-tisense primer, then a nested PCR was performed using the reversed transcripted cDNA as the template. PCR products were cloned into the pMD18 - T vectors, followed by sequencing. Results Specific fragments were amplified in clinical samples of SARS patients and confirmed by DNA cloning and sequencing. Result of blast showed only one nucleic acid different from TOR2 strain of SARS - associated coronavirus. Conclusion Sequencing confirm the existence of SARS - associated coronavirus sequence. Nest RT - PCR could be a quick, easy, and convenient way of detecting SARS- associated coronavirus.