Campbell G R, Prosser J, Glover A, Killham K
Department of Plant and Soil Science, University of Aberdeen, Aberdeen, Scotland, UK.
J Appl Microbiol. 2001 Dec;91(6):1004-10. doi: 10.1046/j.1365-2672.2001.01465.x.
To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water.
Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration.
A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water.
The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.
评估基于多重PCR的检测方法用于灵敏快速检测土壤和水中大肠杆菌O157:H7的适用性。
在土壤和水样中加入大肠杆菌O157:H7，在进行多重PCR之前进行两个阶段的富集培养。检测灵敏度高达饮用水中1 cfu/ml和土壤中2 cfu/g。在将大肠杆菌O157:H7加入土壤之前使其饥饿35天，并不影响该检测方法检测低至10 cfu/g土壤中初始细胞数的能力。采用8小时的初次富集培养能够检测低至6 cfu/g土壤中的大肠杆菌O157:H7，采用6小时的初次富集培养则能检测10⁴ cfu/g土壤中的该菌。当向土壤中接种10⁵ cfu/g大肠杆菌O157:H7时，PCR检测显示在35天的培养期间该菌持续存在。然而，当向土壤中接种较低数量的病原菌时，PCR扩增信号表明其存活情况取决于细胞浓度。
基于多重PCR的检测方法与富集策略相结合，能够灵敏快速地检测土壤和水中的大肠杆菌O157:H7。
在一个工作日内灵敏检测环境材料中大肠杆菌O157:H7的能力相较于检测该病原菌的其他耗时更长的方法有了显著进步。
