广东省传染性非典型肺炎病例的SARS冠状病毒分离株的分子生物学鉴定

Molecularbiology identification of SARS coronavirus from SARS patients in Guangdong

目的 对广东省部分传染性非典型肺炎 (SARS)病例的SARS冠状病毒分离株进行鉴定 ,并初步建立SARS冠状病毒PCR检测方法。方法 采集临床诊断为SARS病例的咽漱液标本 ,进行多种细胞分离培养 ,对出现病变的细胞分离物采用冠状病毒特异引物通过逆转录聚合酶链反应 (RT PCR)及巢式聚合酶链反应 (Nested PCR)进行扩增 ,并对部分扩增片段进行序列测定 ,应用DNASTAR软件分析比较。同时采用间接免疫荧光 (IFA)和ELISA法检测上述患者血清中SARS冠状病毒IgG抗体。结果 对 2 1例SARS患者的细胞分离物进行PCR扩增 ,结果均阳性 ,其中 1 2份患者咽漱液标本PCR扩增结果也阳性 ,序列测定及基因分析表明 ,广东省SARS病例的病毒分离株为冠状病毒 ,其基因序列与WHO公布的SARS基因序列一致 ,氨基酸序列与目前已知的冠状病毒同源性为 5 8%～ 76 %。同时对 2 1例患者进行血清学检测 ,其中IFA检测有 9例阳性 ,ELISA检测有 1 2例阳性。结论 从广东省部分SARS病例标本中分离的病毒株是一种新的冠状病毒 ,PCR检测具有早期。

Objective To identify the isolates from patients with SARS in Guangdong and to develop a rapid diagnostic technique. Methods Throat washings collected from patients with SARS were inoculated into several cell lines. The isolates were analyzed by reverse transcript PCR, nested PCR, sequence, and DNASTAR software respectively. IFA and ELISA were applied to detect IgG of SARS virus simultaneously. Results A total of 21 isolates were positive by PCR amplification, and 12 throat washings were positive too by PCR amplification. The data of sequencing show that the isolates from patients were coronaviruses, and the nucleotide sequence was the same as SARS virus published by WHO, the deduced amino acid sequence had 58%～76%homogeneity compared with other known coronaviruses. The sera of 21 patients were tested by IFA and ELISA respectively, 9 positive by IFA and 12 positive by ELISA. Conclusion The strain isolated from some patients with SARS was a novel corona virus which could be tested by PCR rapidly and promptly.