RT-SHIV rt基因单拷贝PCR扩增方法的建立及初步应用

摘要

Objective To establish a single genome amplification assay for obtaining complete rt gene sequence from RT-SHIV chimeric virus. Method Specific primers were designed with Oligo and screened through serial dilution, then PCR was optimized to amplify complete rt gene by selecting the best annealing temperature and cycles. Serial dilutions of RT-SHIV chimeric plasmid was used as template standards to establish this system. Complete rt gene of infected monkey in vivo were amplified using this method, and the obtained sequences were analyzed with BioEdit software. Results A group of nested PCR primers was got and a single genome amplification assay was established to obtain complete rt gene. Single genome sequences were obtained using dilution of 100 copies/μL plasmid. The results show that the RT-SHIV-infected monkeys had one and six amino acid variations at d266 and d294. Conclusions The single genome amplification assay established in this study is an highly sensitive, speeifie and reprodueible method. It ean be used to analyze eomplete rt gene from various types of RT-SHIV.%目的 建立RT-SHIV病毒全长rt基因单拷贝PCR扩增方法,用于HIV-1 rt基因体内遗传与变异研究.方法 Oligo软件设计RT-SHIV rt基因特异性扩增引物,梯度稀释方法进行特异性和灵敏度筛选,进而优化退火温度和PCR反应最佳循环数等条件,建立rt基因PCR扩增方法;在此基础上将模板进行有限稀释,摸索rt基因单拷贝PCR扩增条件;使用该方法扩增感染猴体内RT-SHIV病毒rt基因,BioEdit软件进行基因序列分析.结果 筛选得到一组巢式PCR引物,成功建立了RT-SHIV rt基因PCR扩增方法;当模板浓度为100 copies/μL时,扩增产物为单拷贝序列;测序结果显示RT-SHIV感染猴d266和d294血浆样本分别存在1处和6处氨基酸突变.结论 本研究建立的全长rt基因单拷贝PCR扩增方法特异性好、灵敏度高、重复性强,可以应用于各类RT-SHIV病毒的全长rt基因分析.