新疆农业科学2018，Vol.55Issue(3)：572-580,9.DOI:10.6048/j.issn.1001-4330.2018.03.021
常规PCR法扩增口蹄疫病毒基因组5'端序列的探索
Exploration of Foot-and-Mouth Disease Virus Genome's 5'-end Sequences Amplification by Conventional PCR Method
摘要
Abstract
[Objective] To amplify the 5'-end sequences of the FMDV genome.[Method]RNA frozen in liquid nitrogen of FMDV O/Akesu/58 CE39A strain was reverse-transcribed to cDNA, which was ampli-fied, cloned and sequenced for the 5'terminal sequences.[Result]With the cDNA1 and cDNA3 as templates, LA Taq DNA polymerase, EX Taq DNA polymerase, PrimeSTAR? HS DNA polymerase, and three pairs of primersⅠ,ⅡandⅢwere used to amplify the 5'end fragment, but failed. Only when LA Taq DNA polymer-ase were used as the amplification enzyme, the cDNA2 as the template, and two pairs of primers Ⅰ, Ⅱ the expected fragment of the FMDV 5'terminal fragment could be amplified. And the tested sequences length were 787 bp and 797 bp, homology analysis of nucleotide sequences were 86. 2％ and 86. 3％ between the deter-mined and reference sequences.[Conclusion]Compared with other methods, the conventioanl PCR method possesses the advantages of low cost and simple operation in spite of its low efficiency. In this experiment, 72 PCR was carried out, and the success rate was 6/72, which indicated that the amplification of FMDV 5'termi-nal sequences was difficult, and the inversion of the primers and amplification primers should be rationally de-signed . The experiment has provided an alternative for the research of reverse genetics of a lot of RNA viruses.关键词
口蹄疫病毒/常规PCR/5'端序列/基因扩增Key words
FMDV/conventional PCR/5'-UTR sequences/amplification分类
农业科技引用本文复制引用
朱研,苗书魁,马文戈,李金娜,魏玉荣,汪萍,魏婕,米晓云,黄炯..常规PCR法扩增口蹄疫病毒基因组5'端序列的探索[J].新疆农业科学,2018,55(3):572-580,9.基金项目
"十二五"国家科技支撑计划项目"野生动物跨境传播外来动物疫病监测与控制技术研究"(2013BAD12B04) （2013BAD12B04）
新疆维吾尔自治区科研机构创新发展专项资金项目"口蹄疫疫苗O型种毒质量优化"(2016D4008) （2016D4008）
National Science and Technology Support Program during the 12th Five-Year Plan Period"Study on Surveillance and Control Tech-nology of Exotic Animal Disease Transmitted by Wild Animals across the Border" (2013BAD12B04) and Innovative Development Special Fund Project of Scientific Research Institutions of Xinjiang Uygur Autonomous Region"Quality Optimization of Foot-and-mouth Disease Vaccine Type O Virus" (2016D4008) （2013BAD12B04）
