肾综合征出血热病毒逆转录－聚合酶链反应检测方法的实验研究和临床意义

Experimental Study on Reverse Transcription-Polymerase Chain Reaction for Detection of Hemorrhagic Fever with Renal Syndrome virus and its Clinical Significance

选择覆盖肾综合征出血热病毒（ＨＦＲＳＶ）核蛋白（ＮＰ）基因的保守区核苷酸序列合成引物，采用异硫氰酸胍一步法抽提ＲＮＡ，建立了逆转录（ＲＴ）聚合酶链反应（ＰＣＲ）检测临床血清样本中ＨＦＲＳＶＲＮＡ的方法。扩增产物经凝胶电泳及Ｓｏｕｔｈｅｒｎ印迹杂交证实了其具有特异性。结果显示ＨＦＲＳＶ阳性细胞培养液、１６份患者血清均为阳性，而１０份正常人血清均为阴性。该方法具高特异、高敏感的特点，为临床早期、快速诊断，并从分子生物学角度鉴定ＨＦＲＳＶ提供了新手段。

Hemorrhagic fever with renal syndrome virus(HFRSV) has a tripartite, single-stranded, negative-sense RNA genome.Two 20-mer oligonucleotides flanking the s segment were chosen as primers. RNA, extracted by single-step method of acid guanidinium thiocyanate-phenol-chloroform; A reverse transcription-polymerase chain reaction(RT-PCR) was adopted for detection of HFRSV in viral infected Vero-E6 cells and in clinical serum speimens. The PCR product was then detected by agarose gal electrophoresis and southern hybridization. Serum samples from all the sixteen patients showed positive result.while the normal serum samples showed negtive result. It was suggested that the detection method of RT-PCR is simple, sensitive, specific and can be applied for clinical detection.