霍乱弧菌及肠出血性大肠杆菌多重PCR检测方法的建立

针对霍乱弧菌肠毒素和肠出血性大肠杆菌志贺毒素基因（ctx、stx1和stx2）设计引物,扩增大小不同的特异性片段,优化反应条件,建立多重PCR方法,对人工污染的水样品进行模拟检测。结果显示,多重PCR扩增系统针对目的菌具有高度的特异性。敏感性试验证实,当多重PCR反应体系中模板含量在10CFU时仍能检出。模拟水样品多重PCR检测的敏感性试验证明,人工污染的河水直接集菌检测敏感性为100～1 000CFU/mL,增菌培养后多重PCR检测敏感性可达1CFU/mL。本试验于同一PCR体系检测霍乱弧菌和肠出血性大肠杆菌的毒素基因,可用于这2种致腹泻性病原菌的快速检测。

Development of multiplex PCR for detection of Vibrio cholerae and enterohemorrhagic Escherichia coli

A multiplex PCR method was developed for the rapid detection of genes encoding Shiga toxins 1 and 2（stx1 and stx2） of enterohemorrhagic Escherichia coli（EHEC） and Cholera toxin gene（ctx） of Vibrio cholerae.Three pairs of primers were synthesized.The size of amplified products is different,so it is easy to distinguish the bands on agarose gel.The method includes enrichment before the PCR reaction.The specificity of the multiplex PCR method was determined by using 25 strains of pure-cultured bacteria,including Vibrio cholerae O1 and O139 and 4 EHEC strains.All 6 EHEC strains and Vibrio cholerae strains produced positive bands,whereas the others did not.The sensitivity was evaluated by detection of artificially contaminated samples,with a range of 100-1 000 CFU/mL in contaminated water samples and 1 CFU/mL after enrichment.These results indicated that the Multi-PCR method exhibited high specificity and sensitivity in the detection of EHEC and Vibrio cholerae.It could be used to detec environment samples contaminated by EHEC and Vibrio cholerae.