聚合酶链反应检测汉坦病毒感染的实验研究

Study on the detection of Hantaan virus RNA by RT nested PCR

目的 早期诊断汉坦病毒感染。方法 根据国际标准株（７６１１８ ，８０３９） Ｍ 基因Ｇ１ 区设计两对公有引物和一条探针，按异硫酸氰胍—酚一步法提取汉坦病毒ＲＮＡ，采用ＲＴｎｅｓｔｅｄ ＰＣＲ检测病毒细胞培养上清原液及稀释液、１６ 份对照组血清、４０ 份ＨＦＲＳ急性期（１ ～５ 天） 血清标本中的汉坦病毒ＲＡＮ，所有扩增产物采用Ｓｏｕｔｈｅｒｎ ｂｌｏｔ 或Ｄｏｔ ｂｌｏｔ 证实其特异性。结果 汉坦病毒细胞培养液和４０ 份ＨＦＲＳ急性期血清标本中的３８ 份均可扩增出特异性的片段４５３ｂｐ ，而１０ 份正常人血清和１２ 份非ＨＦＲＳ血清均为阴性，该方法敏感度高于细胞培养。结论 ＲＴｎｅｓｔｅｄ ＰＣＲ 是一种敏感、特异、快速的方法，可用于汉坦病毒感染的早期诊断。

Objective To diagnose the infection of Hantaan virus early.Method According to the published sequence of the M gene G\-1 segments of Hantavirus strain 76 118 and Seoul virus strain 80 39,we designed two sets of genus sepcial primer and a probe.Hantaan virus RNA was extracted using a guanidine isothiocyanate procedure.RT nested PCR was used to amplify the special frament from Hantaan virus cell culture supernatant,16 negtive control samples,40 HFRS serum samples of acute stage(1 5d) patients.All amplified products were confirmed by Southern Blot or Dot Blot.Results The 453bp sequence of Hantaan virus M gene G\-1 was specially amplified from Hantaan virus cell culture supermatant.Hantaan virus RNA was also detected in 38 of 40 HFRS acute stage serum samples.No positive results were found by PCR while detecting 10 normal human serum samples and 12 non HFRS patients serum samples.The sensitivity of RT nested PCR in detecting Hantaan virus infection is superior to that of virus cell culture.Conclusion These results indicate that RT nested PCR is a special and sensitive method in diagnosing the infection of Hantaan virus early.(Shanghai Med J,1999,22:692 694)