套式RT-PCR检测肾综合征出血热病毒方法的建立及应用

ESTABLISHING AND DEVELOPING OF METHODS USING RTPCR DETECT HFRS VIRUSE

针对汉坦病毒（Ｈａｎｔａｎｖｉｒｕｓ，ＨＶ）中国代表株（Ａ９、Ｒ２２）Ｍ基因片段设计分型引物（Ⅰ、Ⅱ型），采用改进的异硫氰酸胍酚一步法（胍酚法）提取汉坦病毒ＲＮＡ，套式ＲＴ－ＰＣＲ检测稀释病毒上清，模拟病人血清和２０例急性期（１～７ｄ）血清样本。结果：能检测到模拟血清中少至几个ＰＦＵ病毒ＲＮＡ，敏感性５～１０ｆｇ。检测过程可在５ｈ内完成。２０例急性期患者血清检测阳性率为１００％，并能进行临床分型。扩增产物经打点杂交证实其特异性。而正常及非ＨＦＲＳ患者血清结果均为阴性。提示：套式ＲＴ－ＰＣＲ基因诊断技术优于传统的ＭａｃＥＬＩＳＡ、ＩＦＡＴ和ＲＰＨＩ诊断法。

To establish gene diagnosis technique of HFRS.we designed typing primer (type ,)according to the sequences of M gene segments of chinese representative stains (A9,R22) of HV.We used improved isothiocyanate guanidiumphenol one step method (guanidiumphenol method )to extract HVRNA and nest RTPCR ,in order to detect various plaque titer viral supernate and imitate patient serum and 20 acute stage (1￣7d) patient serum samples.The results showed that this method could detect several PFU viral RNA a few in imitate patient serum.Sensitivity was 5￣10fg.All process was in 5 hour.primary positive rate was 100% detective to 20 acute stage serum indicated that(7d) and clinical type was done.Specificity of all amplified products was confirmed by dot blotting.No positive result was found by PCR to amplify 20 cases normal human serum and 24 nonHFRS patients serum. Results above indicate that nest RTPCR technique has more advantages than traditional diagnostic techniques of HFRS such as MacELISAIFAT and RPHI.FL)]