Ibekwe A Mark, Watt Pamela M, Grieve Catherine M, Sharma Vijay K, Lyons Steven R
George E. Brown Jr. Salinity Laboratory, U.S. Department of Agriculture-Agricultural Research Service, Riverside, California 92507, USA.
Appl Environ Microbiol. 2002 Oct;68(10):4853-62. doi: 10.1128/AEM.68.10.4853-4862.2002.
Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C(T)) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C(T) and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 x 10(-5) pg of E. coli O157:H7 DNA ml(-1) equivalent to approximately 6.4 x 10(3) CFU of E. coli O157:H7 ml(-1) based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >/=3.5 x 10(4) CFU g(-1). E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g(-1) with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.
在美国的许多大都市地区，地表水和地下水一直被用作饮用水源。由于城市和农业径流中诸如大肠杆菌等污染物的增加，这些水源的水质可能会下降。在本研究中，一种多重荧光定量PCR检测方法被用于定量人工湿地中土壤、粪便、奶牛和小牛粪便以及乳品废水中的大肠杆菌O157:H7。设计引物和探针以便在单一反应中扩增和定量大肠杆菌O157:H7的志贺样毒素1（stx1）和2（stx2）基因以及紧密黏附素（eae）基因。用来自33株带有和不带有这三个基因的大肠杆菌O157:H7及相关菌株的DNA证实了引物特异性。确定了荧光阈值循环（C(T)）与大肠杆菌O157:H7 DNA起始量之间的直接相关性。在PCR检测中使用的每毫升CFU数量与C(T)之间也观察到了类似的相关性。基于平板计数确定检测限为每毫升7.9×10(-5) pg大肠杆菌O157:H7 DNA，相当于每毫升约6.4×10(3) CFU大肠杆菌O157:H7。当细胞数量≥3.5×10(4) CFU g(-1)时，能够对土壤、粪便、粪便和废水中的大肠杆菌O157:H7进行定量。湿地样品中检测到的大肠杆菌O157:H7水平在湿地进水和出水之间下降了约2个对数级。通过16小时的富集，该检测方法在土壤中的检测限提高到小于10 CFU g(-1)。这些结果表明，所开发的PCR检测方法适用于环境样品中大肠杆菌O157:H7的定量测定，代表了不同生态系统中病原体定量方面的重大进展。
