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[目的]建立霍乱毒素和耐热直接溶血素基因双重TaqMan实时-PCR实验室检测方法。[方法]根据霍乱毒素基因(Cholera toxin gene,ctx)和耐热直接溶血素基因(thermostable direct hemolysin,tdh)的保守序列设计引物和TaqMan探针,建立检测霍乱毒素和耐热直接溶血素两种毒力基因的双重TaqMan实时PCR方法。对所建立的霍乱毒素基因和耐热直接溶血素基因双重TaqMan实时PCR检测方法进行灵敏度和特异度评价。[结果]建立了霍乱毒素基因和耐热直接溶血素基因双重TaqMan实时PCR的实验室检测方法。优化的tdh和ct双重TaqMan实时PCR反应体系中,ct引物和探针的浓度分别为200nmol/L和100nmol/L;tdh引物和探针的浓度分别为200nmol/L和100nmol/L。反应体系的灵敏度和特异度均为100%。优化的反应体系对两种质粒模板的检测下限均为1.0×102拷贝/μl,扩增效率分别为94%和97.7%。[结论]本研究建立了基于TaqMan探针的tdh和ct双重实时PCR检测方法,具有令人满意的灵敏度和特异度。检测下限能达到1.0×102拷贝/μl,高于普通PCR100倍。并且双重实时PCR能够在一个反应体系中同时检测两种毒力基因,这为费时又繁琐的传统检测方法提供了一种可靠又快速的替代选择。
[Objective] To establish a dual TaqMan real-time PCR assay for cholera toxin and heat-resistant hemolysin gene. [Method] Primers and TaqMan probes were designed according to the conserved sequence of Cholera toxin gene (ctx) and thermostable direct hemolysin (tdh), and the detection of both cholera toxin and heat resistant hemolysin Dual TaqMan real-time PCR method for virulence genes. The sensitivity and specificity of the double TaqMan real-time PCR assay for cholera toxin gene and heat-resistant direct hemolysin gene were evaluated. [Result] The laboratory test method of double TaqMan real-time PCR of cholera toxin gene and heat-resistant direct hemolysin gene was established. Optimized tdh and ct double TaqMan real-time PCR reaction system, ct primer and probe concentrations were 200nmol / L and 100nmol / L; tdh primer and probe concentrations were 200nmol / L and 100nmol / L. The sensitivity and specificity of the reaction system were 100%. The detection limit of the optimized reaction system for both plasmid templates was 1.0 × 102 copies / μl, and the amplification efficiency was 94% and 97.7%, respectively. [Conclusion] This study established tdh and ct double real-time PCR detection methods based on TaqMan probe with satisfactory sensitivity and specificity. Detection limit can reach 1.0 × 102 copies / μl, higher than the normal PCR100 times. And double real-time PCR can detect two virulence genes in one reaction system at the same time, which provides a reliable and fast alternative to the time-consuming and cumbersome traditional detection methods.