O型口蹄疫病毒3D基因荧光定量PCR方法的建立及应用

Establishment and Application of Fluorescence Quantitative PCR of Type O Foot-and-mouth Disease Virus 3D Gene

为建立快速检测抗FMDV转基因猪中O型口蹄疫病毒(foot-and-mouth disease virus,FMDV)的荧光定量PCR方法,本试验选择FMDV结构蛋白基因3 D的核苷酸序列设计特异性引物,经过RNA抽提、反转录成cDNA、PCR扩增,扩增出了大小为180bp的基因片段。将回收的目的片段与pMD18-T克隆载体连接,转化大肠杆菌DH5α感受态细胞。提取的质粒经PCR、酶切和测序鉴定,证实为FMDV的阳性重组质粒。将验证正确的重组质粒10倍梯度稀释后作模板,进行荧光定量PCR试验,建立FMDV 3 D基因的标准曲线及其回归方程,并确定扩增的最佳退火温度。试验结果显示标准曲线的回归方程为Y=-3.727X+32.04,回归系数R2=0.980,熔解曲线为单一峰。建立的荧光定量PCR方法的灵敏度为1.2×101拷贝/μL,只与FMDV反应。结果表明,建立的荧光定量PCR方法具有特异性强、灵敏度高等特点,为检测FMDV在动物组织细胞中的含量提供了技术方法。

To establish a rapid detection of type O foot-and-mouth disease virus(FMDV)fluorescence quantitative PCR method,we selected FMDV structural protein gene 3 Dto 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 DH5αcells.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 3Dand 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 1.2×101copies/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.