Centre for Environment, Fisheries and Aquaculture Science, Weymouth Laboratory, Weymouth, Dorset, UK.
National Oceanography Centre, Southampton, Hampshire, UK.
Water Res. 2017 Dec 1;126:101-110. doi: 10.1016/j.watres.2017.08.032. Epub 2017 Aug 25.
The presence of Escherichia coli in environmental waters is considered as evidence of faecal contamination and is therefore commonly used as an indicator in both water quality and food safety analysis. The long period of time between sample collection and obtaining results from existing culture based methods means that contamination events may already impact public health by the time they are detected. The adoption of molecular based methods for E. coli could significantly reduce the time to detection. A new quantitative real-time PCR (qPCR) assay was developed to detect the ybbW gene sequence, which was found to be 100% exclusive and inclusive (specific and sensitive) for E. coli and directly compared for its ability to quantify E. coli in environmental waters against colony counts, quantitative real-time NASBA (qNASBA) targeting clpB and qPCR targeting uidA. Of the 87 E. coli strains tested, 100% were found to be ybbW positive, 94.2% were culture positive, 100% were clpB positive and 98.9% were uidA positive. The qPCR assays had a linear range of quantification over several orders of magnitude, and had high amplification efficiencies when using single isolates as a template. This compared favourably with qNASBA which showed poor linearity and amplification efficiency. When the assays were applied to environmental water samples, qNASBA was unable to reliably quantify E. coli while both qPCR assays were capable of predicting E. coli concentrations in environmental waters. This study highlights the inability of qNASBA targeting mRNA to quantify E. coli in environmental waters, and presents the first E. coli qPCR assay with 100% target exclusivity. The application of a highly exclusive and inclusive qPCR assay has the potential to allow water quality managers to reliably and rapidly detect and quantify E. coli and therefore take appropriate measures to reduce the risk to public health posed by faecal contamination.
大肠杆菌存在于环境水中被认为是粪便污染的证据，因此常被用作水质和食品安全分析中的指标。现有的基于培养的方法从采集样本到获得结果需要很长时间，这意味着在检测到污染事件时，它可能已经对公众健康造成了影响。采用基于分子的方法检测大肠杆菌可以显著缩短检测时间。开发了一种新的定量实时 PCR（qPCR）检测方法来检测 ybbW 基因序列，该方法被发现对大肠杆菌具有 100%的排他性和包容性（特异性和敏感性），并直接比较其在环境水中定量检测大肠杆菌的能力与菌落计数、针对 clpB 的定量实时 NASBA（qNASBA）和针对 uidA 的 qPCR。在所测试的 87 株大肠杆菌菌株中，发现 100%的菌株 ybbW 阳性，94.2%的菌株为培养阳性，100%的菌株 clpB 阳性，98.9%的菌株 uidA 阳性。qPCR 检测法在几个数量级上具有定量的线性范围，并且当使用单一致病体作为模板时，具有高扩增效率。这与 qNASBA 相比具有优势，后者显示出较差的线性度和扩增效率。当将这些检测方法应用于环境水样时，qNASBA 无法可靠地定量检测大肠杆菌，而两种 qPCR 检测法都能够预测环境水中大肠杆菌的浓度。本研究突出了 qNASBA 针对 mRNA 定量检测环境水中大肠杆菌的能力的不足，并提出了第一个具有 100%目标排他性的大肠杆菌 qPCR 检测法。应用高度排他和包容性的 qPCR 检测法有可能使水质管理者能够可靠且快速地检测和定量检测大肠杆菌，从而采取适当措施降低粪便污染对公众健康构成的风险。
