检测SARS冠状病毒的套式RT-PCR方法的建立与初步应用

[目的]建立套式RT-PCR检测SARS冠状病毒的方法。[方法]从广东地区SARS患者尸解肺组织、咽拭子、嗽口水及本实验室分离的SARS冠状病毒（SAPS—CoV）组织培养上清中提取RNA，利用针对SARS冠状病毒多聚酶基因的特异性引物，进行逆转录及套式PCR扩增。扩增出的阳性片段连接入pGEM—T载体中，测序后比较其与其他已知SARS冠状病毒的同源性。[结果]从SARS患者尸解肺组织、咽拭子、嗽口水及SARS冠状病毒组织培养上清中均可扩增出阳性片段，经测序后证实是SARS冠状病毒特有序列。[结论]针对SARS冠状病毒多泵酶基因所建立的套式RT—PCR方法适用于临床标本及组织培养标本中SARS冠状病毒的检测。

THE ESTABLISHMENT AND APPLICATION OF DETECTION OF SARS CORONAVIRUS BY NESTED RT-PCR

[Objective]To establish a RT-PCR method for detection of SARS coronavirus(SARS-CoV).[Methods]The RNAs of the SARS-CoV contained in the lung tissue,throat swabs,gargles of patients with SARS and cell cultures of SARS coronavirus were extracted. RT-PCR was performed using SARS-CoV specific primers to amplify the polymerase gene fragments. The amplicons were cloned into the pGEM-T vectors and then sequenced. All the obtained sequences were compared with the published sequence of SARS-CoV.[Results]Specific amplicons could be amplified from the lung tissue,throat swabs,gargle of the SARS patients,cell cultures of SARS coronavirus. The sequence homology analyses revealed the sequences were specific for the SARS-CoV.[Conclusions]The nested RT-PCR to amplify the polymerase gene fragments can be applied to the detection of SARS-CoV from the clinical samples and cultural samples.