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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 rapid method to identify Chinese measles virus vaccine and wild virus. Methods The Hemagglutinin gene (H gene) was searched for restriction endonuclease sites that could distinguish the Chinese measles virus vaccine strain from the wild-type strain. The gene containing this restriction enzyme site The reverse transcription-polymerase chain reaction (RT-PCR) primers were designed and the sensitivity and specificity of the established RT-PCR method were tested. The restriction of RT-PCR products Restriction Fragment Length Polymorphism (RFLP) was used for restriction analysis. Results The one-step RT-PCR method was sensitive and at least capable of detecting 4.64 TCID50 (50% Tissue Culture Infective Dose) measles virus. No non-measles virus RT-PCR results showed no positive bands, indicating RT-PCR method specific. After the PCR products were digested with Af1Ⅱ, the Chinese measles virus strain Shanghai 191 and the long 47 PCR products could be cut into two fragments, 287bp and 151bp, respectively. The two representative strains of wild measles virus could not be digested with Af1Ⅱ. Conclusion The newly established RT-PCR-RFLP is a rapid and simple method to identify the Chinese measles virus vaccine strains and wild-type strains.