SARS病毒基因的PCR扩增及序列测定

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

目的 利用 RT- PCR技术检测 SARS疑似或临床确诊病人咽拭子、血清及白细胞中的 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 4 0 % ;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 2 2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 Gen Bank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。

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.