SARS冠状病毒多聚酶基因临床检测方法的建立

目的分析SARS冠状病毒广东株多聚酶基因片段的异质性,建立RT-PCR检测SARS冠状病毒的方法,为SARS的快速诊断提供依据。方法合成针对SARS冠状病毒多聚酶基因区段的特异性引物,从广东省SARS病人及疑似病人标本中提取RNA,经反转录和巢氏PCR扩增出相应大小的DNA片段。对这些片段克隆后进行DNA序列分析,并将序列与SARS冠状病毒和其他已知冠状病毒进行同源性分析。结果从多例SARS病人痰、咽拭子或血液标本中得到RT-PCR阳性片段,随机选取其中8例经克隆和DNA序列分析证实均为SARS冠状病毒序列(同源性100%),而所有阴性参照标本RT-PCR均为阴性。克隆片段BNI109与已知冠状病毒在核苷酸和氨基酸水平进行分析显示,该片段与牛冠病毒(bovinecoronavirus,BCV)和鼠肝炎病毒(murinehepatitisvirus,MHV)同源性最高,在氨基酸水平上同源性高达75%。结论SARS冠状病毒多聚酶基因片段BNI109的保守性极强,适合建立RT-PCR法检测SARS冠状病毒。

Clinical detection of polymerase gene of SARS-associated coronavirus

Objective To analyze the heterogeneity of polymerase gene fragment of SARS-associated coronavirus from SARS patients, and establish a RT-PCR method for de tecting SARS-associated coronavirus. Method RT-PCR was performed using SARS co ronavirus-specific primers to amplify the polymerase gene fragment of SARS-assoc iated coronavirus from specimens of suspected and established SARS cases. The a mplicons were cloned and sequenced. All the obtained sequences were compared wi th the sequence of published SARS-associated coronavirus, and alignment was proc eeded with other coronavirus sequences. Results Specific amplicons can be ampli fied from the sputum samples, throat swab and plasma of most SARS patients, and 8 were random selected and sequenced. All of them possessed 100% homology with the published SARS-associated coronavirus sequence, while all the negative cont rols were RT-PCR negative. Nucleotide-sequence and amino acid-sequence alignmen t of the fragment BNI109 with other six known coronavirus show that the fragment BNI109 is more close to bovine coronavirus(BCV) and murine hepatitis virus(MHV ). The BNI109 fragment showed 75% homology with BCV and MHV at amino acid level . Conclusion The polymerase fragment BNI109 of SARS coronavirus is highly cons ervative and is suitable for detecting SARS-associated coronavirus using RT-PCR method.