中国麻疹病毒疫苗株与野毒株鉴别方法的建立

The Establishment of Method for Identifying China Vaccine Strains and Wild Strains of Measles Virus

目的研究建立一种简便快速的鉴别中国麻疹病毒疫苗株与野病毒的方法。方法在血凝素(Hemagglutinin)蛋白基因(H基因)上,寻找能将中国麻疹病毒疫苗株区别于野毒株的限制性内切酶酶切位点,并在包含此酶切位点的基因片段上设计逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)引物,同时对所建立的RT-PCR方法进行敏感性和特异性实验证明,对RT-PCR产物利用限制性片段长度多态性分析方法(Restriction Fragment Length Polymorphism,RFLP)进行酶切鉴定。结果该一步法RT-PCR方法比较敏感,至少能够检测4.64TCID50(50%组织培养感染剂量,Median Tissue Culture Infective Dose)麻疹病毒。而非麻疹病毒RT-PCR结果均未见阳性条带,说明RT-PCR方法特异。PCR产物经Af1Ⅱ酶切作用后,中国麻疹病毒疫苗株沪191与长47PCR产物均能被切成两个片段,分别为287bp和151bp,2株麻疹野病毒代表株均不能被Af1Ⅱ酶切。结论新建立的RT-PCR-RFLP是一种快速、简便的鉴别中国麻疹病毒疫苗株与野毒株的方法。

Objective To establish a simple and quick method for identifying China vaccine strains and wild strains of Measles Virus. Methods To search the enzyme site in Hemagglutinin gene of measles virus for different domestic vaccine strains and wild strains of measles virus,and design the RT-PCR primer within the range covering the enzyme site,and then to confi rm the specifi city and sensibility of the RT-PCR method,and then identify the RT-PCR product by RFLP. Results The one-step RT-PCR method is sensitive,the measles virus of 4.64 TCID50 can be detected at least. No positive bands can be found in the non-measles virus strains,it means that the RT-PCR method has good specifi city,the PCR products of Chian measles vaccine strains of Shang-191 and Chang-47 were all cut into two fragments （287bp and 151bp） by Afi II,but two measles wild virus strains can＇t be cut by A？ II. Conclusion The RT-PCR-RFLP method which we established is a rapid and simple method for identifying China vaccine strain .and wild strain.