摘要
目的 探讨冠状病毒感染与传染性非典型肺炎发病的关系 ,分析冠状病毒基因序列变异的临床意义 ,建立诊断冠状病毒变异株感染的分子生物学方法。方法 收集非典型肺炎 (SARS)患者的不同临床标本 ,采用PCR技术分别进行衣原体、甲肺炎病毒以及冠状病毒等的基因检测。以冠状病毒相对保守的依赖RNA的RNA多聚酶基因为靶基因 ,采用NestedRT PCR技术从非典型肺炎患者的鼻咽吸取物标本和漱喉液标本中扩增出冠状病毒基因 ,将PCR产物直接克隆到T载体 ,进行序列测定 ,进一步比较不同分离株间的核苷酸和氨基酸序列同源性。结果 在 2 7例非典型肺炎患者中检出冠状病毒基因阳性 6例 ,阳性率为 2 2 2 %。而在 15例健康对照中 ,全部阴性。采用BLAST软件将所获得的冠状病毒的基因序列与基因库 (GenBank)登记的序列比较 ,结果显示本次分离克隆的冠状病毒基因与既往报道的所有冠状病毒在核苷酸水平没有相关性 ,核苷酸同源性最高不超过 4 0 %。但在氨基酸水平的同源性为 80 %左右 (70 %～ 82 % )。从不同患者中分离的冠状病毒基因之间在核苷酸水平的同源性在 98%以上。与刚刚公布的加拿大、中国香港、英国等SARS冠状病毒的核苷酸序列同源性在 99%以上。结论 相当部分非典型肺炎患者存在冠状病毒的感染 。
Objective To investigate the etiologic agents of the SARS and develop diagnostic method for this disease Methods Thirty six nasopharyngeal aspirate specimens from 27 patients with SARS in Shenzhen were collected The samples were aliquotted to three parts and subjected to molecular assays for human metapneumovirus, chlamydia and a novel coronavirus, which was reported recently to be the etiologic agent of SARS Nested RT PCR was used to amplify the RNA polymerase gene of the novel coronavirus and the PCR products were sequenced directly or after cloned to pMD18 T vector Results Human metapneumovirus and chlamydia genes were detected in none of the specimens using the RT PCR and nested PCR, respectively The novel coronavirus gene were amplified in 6 of 36 specimens, the sequence analysis indicated that this novel coronavirus is unrelated to any other coronavirus reported previously The nucleotide and deduced amino acid alignment between this coronavirus and others was not more than 40% and 70% to 82%, respectively, while the nucleotide sequence cloned from the 6 patients were identical Conclusion The SARS patients in Shenzhen were infected with coronavirus and this novel coronavirus is associated with SARS The sequence analysis indicated that the coronavirus from SARS patients in Shenzhen is the same as that identified from other areas such as Canada and Hong Kong A specific diagnostic nested RT PCR was developed to identify this novel coronavirus infection
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2003年第2期137-139,共3页
Chinese Journal of Experimental and Clinical Virology
关键词
冠状病毒
基因
克隆
序列
传染性非典型肺炎
病原学
调查
核苷酸
氨基酸
Pneumonia,Viral
Severe Acute Respiratory Syndrome/SARS
Coronavirus OC43, Human
Reverse transcriptase polymerase chain reaction/RT PCR
