SARS病毒基因的PCR扩增及序列测定

目的 利用RT—PCR技术检测SARS疑似或临床确诊病人咽拭子、血清及自细胞中的SARS病毒，并分析了扩增产物内一个可能的突变位点。方法 采用Trizol试剂或病毒RNA提取试剂盒，抽提SARS疑似患者咽拭子、血清及外周血白细胞中的RNA，进行RT—PCR和nest PCR反应，检测SARS病毒RNA，并对扩增产物进行DNA测序。结果 14例SARS疑似患者中2例咽拭子RT—PCR结果阳性，约占14％；5例临床确诊病人中2例咽拭子nest PCR结果阳性，约占40％；6例临床确诊SARS病人中1例血清nest PCR结果阳性，占17％，其他5例疑似患者结果阴性；在22份疑似患者外周血白细胞中未检测到SARS病毒；扩增产物DNA序列与美国和加拿大在GenBank中报道的序列一致。结论 RT—PCR方法是一种快速有效的SARS病毒检测手段，为临床诊断及发病机制研究提供了依据；扩增片段内未发现突变。

Detection of SARS-associated coronavirus in SARS patients by RT-PCR

Objective To detect SARS-associated coronavirus in oropharyngeal swabs,serum and leucocyte samples from suspected or confirmed SARS patients by RT-PCR, and analyse one probable mutation site at position 18282 in the product of RT-PCR. Methods RNA was isolated from clinical specimens(including oropharyngeal swab, serum, leucocytes)by Trizol reagent or QIAamp Viral RNA Mini kit. One-step amplification reactions or nest PCR were performed to detect SARS-associated coronavirus, and nest PCR product was sequenced. Results Viral RNA was detected in 2 of 14 (14%) oropharyngeal swabs from suspected patients by RT-PCR, and 2 of 5 (40%) oropharyngeal swabs from probable patients by nest PCR. Among 11 serum samples, only one was found SARS-associated coronavirus by nest PCR. No viral RNA was detected in 22 leucocyte samples. The sequence of nest PCR product was consistent with that published at GenBank. Conclusion RT-PCR on clinical specimen can confirm the diagnosis of SARS-associated coronavirus infection in suspected patients. No mutation site was found in the nest PCR product.