Office of Water, Technical Support Center, US Environmental Protection Agency, Cincinnati, Ohio 45268, United States.
Environ Sci Technol. 2011 Mar 15;45(6):2250-6. doi: 10.1021/es103365b. Epub 2011 Feb 22.
A sensitive and specific method that also demonstrates viability is of interest for detection of E. coli O157:H7 in drinking water. A combination of culture and qPCR was investigated. Two triplex qPCRs, one from a commercial source and another designed for this study were optimized from 5 different assays to be run on a single qPCR plate. The qPCR assays were specific for 33 E. coli O157:H7 strains tested and detected 500 cells spiked in a background of 10(8) nontarget bacterial cells. The qPCR detection was combined with an enrichment process using Presence Absence (P/A) broth to detect chlorine and starvation stressed cells. qPCR analysis performed post-enrichment allowed the detection of 3-4 cells/L as indicated by a sharp increase in fluorescence (lowering of Ct values) from pre-enrichment levels, demonstrating a 5-6 log increase in the number of cells. When six vulnerable untreated surface water samples were examined, only one was positive for viable E. coli O157:H7 cells. These results suggest that the culture-PCR procedure can be used for rapid detection of E. coli O157:H7 in drinking water.
一种灵敏且特异的方法，同时也能证明其生存能力，对于检测饮用水中的大肠杆菌 O157:H7 具有重要意义。本研究采用了培养法和 qPCR 法相结合的方式。从 5 种不同的检测方法中优化了两种三联体 qPCR，一种来自商业来源，另一种为本研究设计，以便在单个 qPCR 板上运行。qPCR 检测方法对 33 株大肠杆菌 O157:H7 进行了特异性检测，能够在 10^8 个非目标细菌细胞的背景下检测到 500 个细胞的污染。qPCR 检测与利用 Presence Absence（P/A）肉汤进行的富集过程相结合，可以检测到氯和饥饿胁迫的细胞。富集后的 qPCR 分析表明，荧光（Ct 值降低）从预富集水平急剧增加，表明细胞数量增加了 5-6 个对数级，可以检测到 3-4 个细胞/L，这表明存在活的大肠杆菌 O157:H7 细胞。当检测六个脆弱的未经处理的地表水样本时，只有一个样本中存在活的大肠杆菌 O157:H7 细胞。这些结果表明，培养-PCR 程序可用于快速检测饮用水中的大肠杆菌 O157:H7。
