RT—PCR—RFLP用于麻疹病毒疫苗株和H1基因型的分析

Genotype analysis for measles vaccine strain and wild.type viruses of H1 genotype by RT-PCR-RFLP

目的建立RT—PCR—RFLP方法用于天津地区2002--2008年流行的麻疹野病毒基因型研究。方法采集疑似麻疹患者的尿标本和咽拭子，传代细胞分离病毒。提取病毒液中的RNA，用一步RT—PCR法扩增麻疹病毒核蛋白（nucleoprotein，N）基因C端594个核苷酸片段，扩增产物经BcnI酶切后琼脂糖凝胶电泳进行限制性片段多态性分析（RFLP），同时与序列分析进行对比验证。根据结果构建基因系统发生树进行亲缘关系和遗传距离分析。结果2002--2008年189份标本，分离获得69株麻疹病毒，N基因C端RT—PCR检测全部阳性，RFLP分析H1a是优势流行亚型，占98．55％（68／69），仅1株（1．45％）为H1b基因亚型，分型结果与序列测定完全一致。系统发生树分析显示H1a亚型毒株分为2个亚枝，变异范围为0．2％～3．8％，存在不同病毒株引起的传播链共循环。结论基于BcnI限制性内切酶的RT—PCR—RFLP方法能够特异性区分A基因型、H1a、H1b基因亚型，具有快速、简便、准确和经济的优点，更适用于国内大规模麻疹病毒的监测。

Objective To establish RT-PCR-RFLP method for studying the genotype of wild measles virus strains isolated from Tianjin area from 2002 to 2008. Methods Isolations of measles virus were carried out by tissue culture method from urine and throat swab specimens collected from suspected cases. RNA were extracted from the virus specimens. The 594 bp fragment of C terminal of the N （nucleoprotein） gene was amplified by one-step RT-PCR, then the PCR products were digested with Bcn Ⅰ , separated on agarose gel electrophoresis and then analyzed by the method of RFLP （restriction fragment length polymorphism）. In addition, above results were compared with DNA sequencing. Phylogenetic tree was plotted based on the results for the genetic relationship and distance analysis. Results Sixty-nine measles virus strains were isolated from 189 specimens from 2002 to 2008, of which the C terminals of N gene were all detected positive. Among the 69 strains of measles virus isolates, 98.55% （68/69） belonged to Hla sub-genotype which was the predominant sub-genotype, and only one strain （1.45%） belonged to Hlb sub-genotype by RFLP analysis which was in accordance with the results by DNA sequencing method. Phylogenetic tree analysis indicated the Hla sub-genotype measles virus strains should be further divided into 2 clades, and the variation fluctuated between 0.2% and 3.8%. There were transmission chains caused by different virus strains co-circulation. Conclusion A genotype, Hla and Hlb sub-genotype can be identified by RT-PCR- RFLP assay specically based on the restriction enzyme Bcn Ⅰ. The RT-PCR-RFLP assay can be a rapid, simple, accurate and efficient method for large-scale surveillance of measles virus strains in China.