【摘要】 采用异硫氨酸胍-酚-氯仿（AGPC）一步法提取病毒-RNA，并依据肾综合征出血热病毒（HFRSV）核蛋白（NP）编码基因保守区核苷酸序列合成两对巢式引物，建立了逆转录巢式聚合酶链反应（RT－nestedPCR）检测HFRSVRNA方法。应用此法对HFRSV感染的VeroE6细胞培养液及HFRS患者血清中的病毒RNA进行检测。结果显示，感染细胞培养液及35例HFRS患者血清均为阳性，正常的VeroE6细胞及20例正常人血清均为阴性。将HFRSV感染细胞提取的RNA进行10倍系列稀释，可检出10（－7）稀释度的病毒RNA（约0.2pg）。该法灵敏、特异、快速、简便，为从分子水平探讨HFRS的发病机理、临床早期快速诊断提供了新的研究手段。更多还原

【Abstract】 Hantavirus (HV) has a tripartite, single - stranded, negative - sense RNA genome. Two pairs of oligonucleotides flanking S segment were chosen as primers for reverse transcription Polymerase chain reaction(RT - nested PCR). The method of RT - nested PCR was used for detection of virus RNA in HV infected Vero E6 cells and clinical serum specimens from 35 hemorrhagic fever with renal syndrome(HFRS) patients, with HV gene S segment plasmid cDNA as postive control,and virus RNA was extracted by Acid Guanidinium Thiocyanate - Phenol - Chloroform Single- Step Method. The results showed that all the patients’ serum samples and HV infected Vero E6 cells were positive, on the other hand, all the twenty normal serum samples and Vero E6 cells were negative. It suggested that RT - nested PCR was sensitive, specific, rapid and simple, and can be applied for study on the pathogenesis of HFRS in molecular level and early clinical diagnosis.更多还原