Objective To establish a single genome amplification assay for obtaining complete rt gene sequence from RT-SHIV chimeric virus. MethodSpecific 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. ResultsA 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. ConclusionsThe single genome amplification assay established in this study is an highly sensitive, specific and reproducible method. It can be used to analyze complete rt gene from various types of RT-SHIV.