关键词: 口蹄疫病毒; 荧光定量PCR; 重组质粒; 标准曲线

Abstract: To establish a rapid detection of type O foot-and-mouth disease virus (FMDV) fluorescence quantitative PCR method, we selected FMDV structural protein gene 3D to design specific primers, after RNA extraction and PCR amplification, We amplified 180 bp gene fragment. The fragment was purified, cloned into pMD18-T vector, and transformed into competent Escherichia coli DH5α cells. Recombinant plasmid was identified by PCR, enzyme digestion and sequencing, and confirmed that it was the positive recombinant plasmid of FMDV. The recombinant plasmid was gradient diluted by 10 times, we established the standard curve of FMDV structural protein 3D and linear regression equation by fluorescence quantitative PCR method, and determined the optimal amplification temperature. The standard curve was Y=-3.727X+32.04, R²=0.980, and melting curve showed that there was a single peak.The detection limit of fluorescence quantitative PCR method was 1.2×10¹ copies/μL, and could only react with FMDV.These data suggested that the method had strong specificity and high sensitivity, which provided technical methods for the detection of content of FMDV in animal tissues and cells.

Key words: foot-and-mouth disease virus (FMDV); fluorescence quantitative PCR; recombinant plasmid; standard curve