Exploration of Foot-and-Mouth Disease Virus Genome's 5'-end Sequences Amplification by Conventional PCR Method

【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 amplified, 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 primers Ⅰ, Ⅱ and Ⅲ were used to amplify the 5'end fragment, but failed. Only when LA Taq DNA polymerase 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 determined 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' terminal sequences was difficult, and the inversion of the primers and amplification primers should be rationally designed. The experiment has provided an alternative for the research of reverse genetics of a lot of RNA viruses.