限制性片段长度多态性分析方法应用于中国麻疹野病毒基因型别的鉴定

Genotype Identification for Wild-type Measles Virus by Restriction Fragment Length Polymorphism (RFLP)Method

目的 建立和应用麻疹野病毒基因型快速诊断方法 ,及时监测麻疹流行株基因型动态 ,尽早发现异型输入病例。方法 应用一种适用于我国现流行麻疹野病毒的基因型别筛查、定型的分析方法 ,即逆转录 聚合酶链反应 限制性片段长度多态性分析方法 (RT PCR RFLP) ,对吉林省 2 0 0 1～ 2 0 0 3年分离到、经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用脱氧核糖核酸 (DNA)序列分析证实为H1基因型的 9株野病毒进行验证。结果 9株麻疹病毒分离阳性株的RT PCR-RFLP基因分型结果与核酸序列分析结果完全一致 ,均为H1基因型。并应用该方法对吉林省 2 0 0 3年分离的 2株麻疹病毒进行基因型别鉴定 ,亦为H1基因型。同时对 2 0 0 1～ 2 0 0 3年的 46份麻疹病例的临床标本 ,应用新建立的RT-PCR-RFLP方法直接进行麻疹病毒核糖核酸 (RNA)提取、RT-PCR反应及基因型别鉴定。 46例临床标本经直接RT-PCR扩增后的 31例RT-PCR阳性产物 ,经RFLP法酶切、电泳结果均为H1基因型。结论 RFLP分析方法是一种快速、简便又经济实用的中国麻疹野病毒基因定型筛查方法 ,对快速掌握麻疹病毒基因型流行动态及地理分布 ,以及麻疹野病毒的输入、变异情况 。

Objective A simple genotyping method for current wild measles viruses in China. Methods RT-PCR-RFLP method was used.Nine measles viruses,which were isolated during 2001-2003 by our laboratory were identified as H_1 genotype by DNA sequence by national measles laboratory of Chinese Center for Disease Control and Prevention and were tested by this new method. The genotyping results for measles wild viruses by RT-PCR-RFLP were the same with that of DNA sequence:H_1 genotype.We also used this method to identify two measles viruses isolated in 2003 as H_1 genotype.At the same time,we applied this new RT-PCR-RFLP directly to 46 measles clinical samples collected during 2001-2003.Thirtyone of 46 measles clinical samples were RT-PCR positive and then PCR positive products were genotyped by this RFLP method. Results The results of electrophoresis showed that all of them were H_1 genotype. Conclusion The study proved that the RFLP method was rapid, simple, and applicable for genotyping chinese wild measles virus.