核苷酸序列测定法定量评价病毒相对适合度  

Quantitative evaluation of viral fitness by nucleotide sequence determination

作　　者：赵耀[1] 赵晓东[1] 

机构地区：[1]重庆医科大学附属儿童医院P2实验室,重庆400014

出　　处：《重庆医科大学学报》2009年第7期926-929,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金(30471839;30800972)

摘　　要：目的:评价核苷酸序列测定法定量检测病毒相对适合度的可靠性。方法:用RT-PCR法扩增此前筛选获得的Palivizu mab逃逸株F基因片断(分别于F基因第816位和828位带有区别于原型株的遗传标志)。RT-PCR产物经纯化、定量并调节至相同浓度,按不同比例混合后进行核苷酸序列测定。用ABI公司EDIT软件分析序列双峰部位不同RT-PCR产物所占比例,代表病毒适合度水平,用分子克隆法定量验证序列分析定量评估病毒相对适合度的可靠性。结果:按不同比例预混得RSVA2原型株和Palivizumab逃逸株(MP4和F212)分别在F基因第816位和828位出现双峰。峰高测定所获RT-PCR产物比例与预混比例十分接近。采用分子克隆法获得的RT-PCR产物比例亦与序列测定定量分析结果基本一致。结论:序列测定定量分析法评估病毒相对适合度(Relative fitness)结果稳定可靠,简便易行,适用于临床检测HIV等病毒感染性疾病耐药毒株是否出现及其适合度变化。Objective： To evaluate reproductivity of nucleotide sequencing for quantitative determination of viral fitness. Methods： F gene fragments from respiratory syncytial virus （RSV） A2 strain, palivizumab escape mutants, F212 and MP4, were amplified by RT-PCR, gel purified,quantified by spectrophotometry and adjusted to same concentration. A2,F212 and MP4 RT-PCR products were mixed at different ratios and determined for nucleotide sequence. Electropherograms at sites with ambiguity were visually measured by using ABI EDIT software. Proportion of RT-PCR products from prototype virus or escape mutants represented viral fitness level. Molecular cloning was utilized to validate input ratios of RT-PCR products. Results：Dual peaks were seen at 816 and 828 positions in the F gene for A2/F212 and A2/MP4mixtures, respectively. Proportion of prototype virus and escape mutants were gained by measuring the heights of dual peaks. Measured ratios were similar to premixed ratios which were confirmed by molecular cloning. Conclusion： Nucleotide sequencing is reliable, easy assay for evaluation of viral fitness and practical for screening escape mutants and viral fitness in viral infections, such as HIV infection.

关 键 词：呼吸道合胞病毒 适合度 检测手段 核苷酸序列测定 

分 类 号：R373.1[医药卫生—病原生物学]