磁捕法对传染性非典型肺炎冠状病毒RNA富集体系的初步研究

The detection of SARS-associated coronavirus by a specific RNA-capture polymerase chain reaction

目的 初步研究建立磁捕法对传染性非典型肺炎又称严重急性呼吸综合征(SARS)冠状病毒RNA的富集、纯化体系,以提高逆转录聚合酶链反应(RT-PCR)检测SARS冠状病毒RNA的敏感性。方法 根据国际基因库公布的SARS冠状病毒基因序列,自行设计引物、探针,体外克隆目的RNA片段作为标准物质;利用标准RNA以及模拟SARS血清标本的RNA,评价自行设计的SARS冠状病毒巢式RT-PCR检测体系和磁捕法SARS冠状病毒RNA富集检测体系的检测效果。结果SARS冠状病毒巢式RT-PCR检测体系,对于模拟血清SARS标本RNA的最低检测样本浓度为20拷贝/μl,而磁捕法SARS冠状病毒RNA富集检测体系的最低检测样本浓度为1拷贝/μl。结论 初步建立的磁捕法SARS冠状病毒RNA富集检测体系,可有效地对低浓度的RNA样本进行浓缩、纯化,有效提高SARS RT-PCR检测方法的敏感性。

Objective The enrichment for severe acute respiratory syndrome ( SARS)-associated eoronavirus RNA by capturing RNA onto magnetic beads was investigated to improve the sensitivity of reverse transcriptase-polymerase chain reaction (RT-PCR) method. Methods Primers and probes were designed according to the published sequence of the SARS-associated eoronavirus gene. The specific RNA fragment was transcribed in vitro and was quantitied as a standard to evaluate the sensitivity of the detection asssays. Results The minimum concentration of the RNA sample detected by the routine nested RT-PCR assay was 20 copies/μl, however, the minimum concentration of the RNA sample detected by the RNA-capture nested RT-PCR assay was 1 copies/μl. Conclusion The SARS-associated eoronavirus nested RNA-capture RT-PCR method was initially established with higher sensitivity than routine nested RT-PCR method, due to the enrichment for the SARS-associated eoronavirus RNA.