交叉引物扩增技术快速检测淡水产品中四环素耐药基因TetA

Rapid detection of tetracycline resistance gene TetA in freshwater products by cross priming amplification technology

中文关键词:  四环素耐药基因  TetA  交叉引物恒温扩增  快速检测  抗生素耐药

英文关键词:tetracycline resistance gene  TetA  cross priming amplification  rapid detection  antibiotic resistance

中文摘要:

      目的 建立一种基于交叉引物扩增(cross primer amplification, CPA)技术的新型现场快速检测四环素耐药基因TetA的方法。方法 通过特异性设计5条针对TetA基因保守区域的引物, 在单一恒温条件(51~60 ℃)下完成靶标核酸的扩增, 整个检测过程可在45~60 min完成, 且仅需水浴锅或金属浴等基础加热设备。为验证其性能, 研究对方法的灵敏度、特异性及抗干扰能力进行了系统评价。结果 该方法对纯化TetA基因模板的检出限低至30.0 ?fg/μL, 显示出极高的灵敏度。同时, 在与四环素类其他耐药基因(如tetB、tetM)及临床常见病原菌基因的对比测试中, 未出现非特异性扩增, 证实了其优异的选择性。此外, 模拟样本检测结果稳定可靠。结论 本研究成功建立了一种操作简便、快速、低成本且无需复杂仪器的TetA基因CPA检测新方法。该方法具备的高灵敏度与高特异性, 能够有效满足基层医疗机构、养殖场等场所对四环素耐药基因的现场快速筛查需求, 为实时监测和遏制抗生素耐药基因的传播提供了一项有力的技术工具, 具有重要的实际应用价值。

英文摘要:

      Objective To establish a novel method for rapid on-site detection of the tetracycline resistance gene TetA by cross priming amplification (CPA) technology. Methods A total of 5 specific primers targeting conserved regions of the TetA gene were designed. Amplification was performed under isothermal conditions (optimized at 51–60 ℃), with the entire detection process completed within 45–60?minutes using only basic heating equipment such as a water bath or metal bath. To validate its performance, the method was systematically evaluated for sensitivity, specificity and interference resistance. Results The limit of detection for purified TetA gene templates was as low as 30.0? fg/μL, indicating high sensitivity. Furthermore, no nonspecific amplification was observed in comparative tests against other tetracycline resistance genes (e.g., tetB, tetM) and common clinical pathogenic genes, confirming excellent selectivity. Additionally, simulated sample testing yielded stable and reliable results. Conclusion A simple, rapid, low-cost and instrument-free CPA-based method for TetA gene detection is successfully developed. With its high sensitivity and specificity, this method can effectively meet the needs for rapid on-site screening of tetracycline resistance genes in primary healthcare institutions and farming environments. It provides a robust technical tool for real-time monitoring and controlling the spread of antibiotic resistance genes, demonstrating significant practical application value.