【摘要】 [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。更多还原

【Abstract】 [Objective]To develop a duplex TaqMan real-time PCR for the detection of ctx and tdh.[Methods]The conserved region of TDH and CTX gene were used to design primers and probes,and the duplex TaqMan real-time PCR system of detecting ctx and tdh was established.The sensitivity and specificity of duplex TaqMan real-time PCR system was evaluated.[Results]The duplex TaqMan real-time PCR system detecting ctx and tdh was established.In the optimized reaction system of ctx and tdh duplex TaqMan real-time PCR,the concentrations of ctx primers and probe were 200 nmol/L and 100 nmol/L,respectively;the concentrations of tdh primers and probe were 200 nmol/L and 100 nmol/L,respectively.The sensitivity and specificity of this reaction system were both 100%.The detection limits of the optimized reaction system to two plasmids were both 1.0×102 copies/μl,and the amplification efficiency was 94% and 97.7%,respectively.[Conclusion]This study established a duplex TaqMan real-time PCR for the detection of ctx and tdh.This method had satisfying sensitivity and specificity approved by experiment results.The detection limit can be 1.0×102 copies/μl,which is 100times higher than traditional PCR method.Further,duplex real-time PCR can detect two pathogenic bacteria or two virulent genes in the same reaction system at the same time.This method acts as an alternative to the traditional method which is time-consuming and laborious.更多还原