实时荧光定量PCR技术快速检测志贺氏菌

Rapid detection of Shigella by real-time fluorescence quantitative PCR

中文关键词:  志贺氏菌  实时荧光定量PCR  标准曲线  特异性  灵敏性

英文关键词:Shigella  real-time fluorescent quantitative PCR  standard curve  specificity  sensitivity

中文摘要:

      目的 建立实时荧光定量PCR法快速检测志贺氏菌。方法 提取志贺氏菌基因组为模板扩增virA基因片段, 将目的片段连接至pMD19-T载体得到重组质粒, 转化大肠杆菌DH5α感受态细胞, 验证所得到的重组质粒, 以紫外分光光度计测量重组质粒吸光度值A260, 并换算为质粒拷贝数后作梯度稀释得到不同浓度的质粒标准品; 进行荧光定量PCR分析并通过特异性、灵敏性实验以验证该方法的可行性。结果 重组质粒标准品浓度在103~108 copies/μL范围内线性关系良好(r2>0.99), 实时荧光定量PCR检测志贺氏菌时出现良好的扩增曲线, 鼠伤寒沙门氏菌、金黄色葡萄球菌和大肠杆菌均未出现扩增曲线。志贺氏菌的检出限为100 CFU/mL。 结论 本方法便捷、高效、可靠, 可用于志贺氏菌的快速定性定量检测。

英文摘要:

      Objective To establish a method for rapid detection of Shigella by real-time fluorescent quantitative PCR. Methods The Shigella genome was extracted as a template to amplify the virA gene fragment, and the target fragment was ligated into the pMD19-T vector to obtain a recombinant plasmid, which was transformed into Escherichia coli DH5α competent cells. The obtained recombinant plasmid was verified, and the absorbance value A260 of the recombinant plasmid was measured by an ultraviolet spectrophotometer, and it was converted into a plasmid copy number, and then subjected to gradient dilution to obtain plasmid standards of different concentrations. Fluorescence quantitative PCR analysis was performed and the feasibility of the method was verified by specificity and sensitivity experiments. Results The concentration of recombinant plasmid standards within the range of 103?108 copies/μL showed a good linear relationship (r2>0.99). A good amplification curve was observed when real-time PCR was used to detect Shigella. No amplification curves were found for Salmonella typhimurium, Staphylococcus aureus and Escherichia coli. The detection limit of Shigella was 100 CFU/mL. Conclusion The method is convenient, efficient and reliable, it can be used for rapid qualitative and quantitative detection of Shigella.