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目的建立一种灵敏、可靠、快速对水中大肠埃希菌富集和DNA提取的最佳方法组合,以便应用TaqMan探针实时荧光PCR技术进行定量检测。方法膜过滤洗脱环节,采用3种不同的方法进行富集洗脱,通过平板计数法比较各方法富集洗脱效果;DNA提取环节,采用4种方法进行DNA提取。所得DNA进行实时荧光PCR扩增,定量检测DNA拷贝数,比较各方法的DNA提取效率;并采用最优的方法组合,对模拟水样进行膜过滤和DNA提取,考察全过程的回收率和灵敏度。结果采用加表皮葡萄球菌过滤+漩涡混合器+玻璃棒刮擦洗脱方法 (C法),洗脱效率能达到75%~93%;TritonX-100法和磁珠法的线性范围达到6个数量级稀释度(100~10-5),r2分别为0.998和0.999,然而,TritonX-100法更省时,更简便,经济性更好。采用该最优的方法组合,其全过程的回收率为79.7%~104.0%且灵敏度达到1 cfu/ml。结论 C法+Triton-100法组合可以快速、准确、经济地对饮用水中大肠埃希菌进行富集和DNA提取。
Objective To establish a sensitive, reliable and rapid combination of the best method for the enrichment of Escherichia coli in water and DNA extraction for quantitative detection using real-time TaqMan probe real-time PCR. Methods Three different methods were used for enrichment and elution, and the elution effects were elucidated by plate count method. DNA extraction was performed using four different methods. The obtained DNA was amplified by real-time fluorescence PCR, the DNA copy number was quantitatively detected, and the DNA extraction efficiency of each method was compared. The optimal method was used to perform membrane filtration and DNA extraction on the simulated water samples. The recovery rate and sensitivity . Results The efficiency of elution was 75% -93% with the addition of Staphylococcus epidermidis + vortex mixer + glass rod scraping and elution method (C method). The linear range of TritonX-100 method and magnetic bead method reached 6 orders of magnitude dilution Degrees (100 ~ 10-5), r2 respectively 0.998 and 0.999, however, TritonX-100 method is more time-saving, more convenient and more economical. The best combination of the method, the whole process of recovery was 79.7% ~ 104.0% and the sensitivity of 1 cfu / ml. Conclusion Combination of C + Triton-100 method can enrich Escherichia coli in drinking water and extract DNA rapidly, accurately and economically.