单拷贝pcr扩增恒河猴模型体内rt-shiv嵌合-中国比较医学杂志.docVIP

研究报告 RT-SHIV rt基因单拷贝PCR扩增方法的建立及初步应用 鞠 斌，王 卫，丛 喆，姚 南，陈 霆，杜文达，苏爱华，高锡强，魏 强 （中国医学科学院医学实验动物研究所，卫生部人类疾病比较医学重点实验室，国家中医药管理局人类疾病动物模型三级实验室，北京 100021） 【摘要】 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。 【关键词】 RT-SHIV；单拷贝PCR；rt基因 【中图分类号】R33【文献标识码】A【文章编号】1671-7856（2011）10-0000-00 Doi:10.3969/j.issn.1671-7856.2011.10.000 Optimization and establishment of Single Genome Amplification assay using for RT-SHIV rt gene detection JU Bin, WANG Wei, CONG Zhe, YAO Nan, CHEN Ting, DU Wen-da, SU Ai-hua, GAO Xi-qiang, Wei Qiang (Key Laboratory of Human Diseases Comparative Medicine, Ministry of Health; Institute of Medical Laboratory Animal Science, Chinese Academy of Medical Sciences; Key Laboratory of Human Diseases Animal Models, State administration of Traditional Chinese medicine, Beijing 100021, China) 【基金项目】国家科技重大专项课题（2012ZX10001-007-008和2012ZX10004-501）。 【作者简介】鞠斌(1988- )，男，硕士研究生，比较医学专业。 【通讯作者】魏强，教授，博士生导师，研究方向：实验动物病毒学。Email: weiqiang0430@。 [Abstract] 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 graded dilution, then optimized the PCR 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 analyze with BioEdit. Results A group of nested PCR primers was got and established a single genome amplification