【摘要】 研究建立了一种适用于中国现流行麻疹野病毒基因型的基因型别筛查、定型的分析方法 ,即逆转录 聚合酶链反应 限制性内切酶片段长度多态性分析 (RT PCR RFLP)。首先对RT PCR方法的敏感性和特异性进行了实验证明 ,对 1 1株麻疹病毒提取核糖核酸 (RNA) ,逆转录套式PCR扩增后全部出现阳性特异条带。在同样的反应条件下 ,扩增 6种不同基因型麻疹病毒 9株 ,同时又扩增其它非麻疹病毒。结果 9株 6种基因型麻疹病毒RT PCR均呈现阳性条带 ,说明本研究采用的RT PCR方法敏感。而非麻疹病毒RT PCR结果为阴性 ,均未见阳性条带 ,说明该RT PCR方法特异。根据H1 和H2 基因型麻疹病毒分别被不同的限制性内切酶SalⅠ和BamHⅠ切割成不同大小的基因片段 ,建立RFLP快速基因定型方法。之后 ,对建立的RFLP方法的特异性和敏感性进行了实验证明。利用该方法对吉林省连续两年分离到的 9株麻疹野病毒 [已经过中国疾病预防控制中心病毒病预防控制所国家麻疹实验室脱氧核糖核酸 (DNA)序列分析证实为H1 基因型 ]进行验证 ,结果均为H1 基因型麻疹病毒 ,证实该PCR RFLP结果与序列分析结果一致 ,也说明该方法敏感。同时 ,又对 7种不同基因型麻疹病毒应用PCR RFLP方法进行实验 ,结果只有H1 和H2 基因型麻疹病毒被限制性内切酶SalⅠ和BamH?更多还原

【Abstract】 We established a simple genotyping method for current wild measles viruses in China:RT-PCR-RFLP method.Firstly,we demonstrated the sensitivity and specificity of the RT-PCR method:The 11 measles viruses were amplified by RT-PCR and all of them were positive,6 genotypes(9 strains)were amplified by the same reaction condition,at the same time,other non-measles viruses were also amplified.All the 6 genotypes (9 strains)of measles virus showed DNA positive strand,it means this RT-PCR method is sensitive for measles viruses. While the RT-PCR result of all the non-measles viruses showed negative DNA strand,it means the RT-PCR method is special for measles viruses,not others.The RFLP genotyping method was established according to the different gene fragment size of H 1 and H 2 genotype measles virus which were cut by different enzyme Sal Ⅰ.And then,we demonstrated the sensitivity and specificity of the established RFLP method:we tested 9 measles viruses isolated by our CDC (all the 9 measles vruses wiere identified as H 1 genotype by DNA sequence by national measles lab.),and the results were also H 1 genotype.It was identified that the result of PCR-RFLP was the same with that of the DNA sequence.It means this RFLP method is sensitive. We also tested 6 genotypes measles viruses by this RFLP method:only H 1 and H 2 genotype were cut as 2 different gene fragments by restriction enzyme SalⅠ and BamHⅠ, it means our RFLP method is special for current strains of Chinese measles viruses.It was concluded that this RFLP method is a rapid,simple,and applicable genotyping method.更多还原