RT-PCR定量检测汉坦病毒RNA及在临床诊断中的价值

Quantitative determination of Hantavirus mRNA with RT-PCR and its value in clinical diagnosis

目的 建立敏感特异的检测肾综合征出血热（HFRS）病人血清中汉坦病毒RNA的定量RT-PCR方法。探讨HFRS患者血清中病毒RNA持续时间，水平及其病情严重程度的关系。方法应用套式RT-PCR扩增引物，自行设计和制备标准品获得标准曲线，使用实时荧光定量PCR技术检测了HFRS（SEO型）血清中的汉坦病毒的载量。结果14例HFRS病人血清中，SEO RNA阳性11例，SEO型病人的病毒载量一般可达10^2～10^4 copies／ml。其中2例发病第8天，2例第9天的病人血清PCR结果仍为阳性。结论：我们采用的实时荧光定量PCR方法可很好的反映HFRS病人血清中汉坦病毒RNA含量。HFRS病人病毒血症持续时间可超过1周，血清中RNA水平与病情严重程度有关。

To establish a sensitive and specific method of assay for quantitative detection of Hantavirus RNA in serum of patients with hemorrhagic fever with renal syndrome （PIFRS） by means of RT-PCR in order to understand the persistence of viruses and its level in patient＇s sera and the relationship of severity of the clinical conditions with serum RNA, the primers were amplified with nested RT-PCR, the standard curve for assay was obtained through the self-designed and prepared standard product, and the load of Hantavirus in patient＇s sera was determined by real-time fluorescence quantitative PCR. It was found that the RNAs of Hantavirus subtype SEO cotdd be detected in 11 out of 14 patients with PIFRS, and the viral RNA copy number was ranged from 10^2 to 10^4 copies/rrd. The detection of viruses cotdd persist up to 8 days or to 9 days after onset of disease, observed both in two cases. It is evident that the real-time fluorescence quantitative PCR assay employed in the present study can reflect definitely the viral load of Hantavirus in sera of patients with PIFRS with a duration of viral persistence for more than one week and a correlation between the serum RNA level and the severity of clinical conditions.