RT-PCR结合限制性片段长度多态性分析对我国汉坦病毒进行基因分型的研究

Study on genotyping of Hantavirus isolates of China by RT PCR/RFLP

用电脑软件分析ＨＦＲＳＶ 囊膜糖蛋白编码基因的Ｍ 节段，寻找不同型别间病毒株Ｍ 节段的限制性内切酶酶切位点的差异。结果发现，ＨＴＮ 型７６１１８ 株和ＳＥＯ 型Ｒ２２ 株Ｍ 节段１１９９～１４９７ 间的酶切位点，可用于进行限制性内切酶基因多态性分析，以确定ＨＦＲＳＶ 的型别。根据上述结果，合成一对引物，建立ＲＴ／ＰＣＲ 方法，分别用Ｒｓａｌ、Ｔａｑ １ 和ＨｉｎｄＩＩＩ３ 种内切酶对扩增片段进行酶切分型。用此方法分析了从我国不同地区、不同宿主分离的毒株１９ 株，及国际标准毒株１ 株。结果表明，９ 株可定为ＨＴＮ 型，８ 株可定为ＳＥＯ 型，３ 株无法确定其型别。此２０ 个毒株曾用血清学方法分型，仅１１ 株分型成功。分型成功率ＲＴＰＣＲ／ＲＦＬＰ法与血清学方法分别为８５％ 和５５％ ，前者比后者高３０ ％ 。

The sequences of M segment genes of the prototype viruses of HTN and SEO serotypes were analysed, which indicated that there were different restriction endonucleas sites in sequence 1199～1497 which could be used for typing The present study reported that 20 Hantanvirus strains were typed by reverse transcriptional polymerase chain reaction(RT PCR) and restriction fragment length polymorphism(RFLP)analysis The results showed that of the 20 Hantanvirus strains, 9 and 8 strains were HTN and SEO, respectively, and the other 3 strains were unable to be determined The experiment suggested that the RT PCR/RFLP could be used for genomic typing in Hantanvirus successfully, while only 11 out of the 20 isolates could be typed by serological methods previously, and which showed that the typing rate by RT-PCR/RFLP and serological method were 85%and 55%, respectively, and the typing rate of the former was higher than that of the latter by 30%