Multiplex polymerase chain reaction method discriminating Escherichia coli and Shigella sp.
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ORIGINAL PAPER
Multiplex polymerase chain reaction method discriminating Escherichia coli and Shigella sp. Y. Yamazaki • A. Fukasawa
Received: 5 June 2010 / Revised: 13 September 2010 / Accepted: 19 October 2010 / Published online: 9 November 2010 Ó Springer-Verlag 2010
Abstract To distinguish between Escherichia coli and other bacteria that have similar biochemical characteristics, 3 polymerase chain reaction techniques were combined. The primer sets cydA-F2-A2 and cydA-R2-A2 were designed to amplify 605 base pairs of nucleotide sequence specific for the cydA gene of Escherichia coli; primer sets lacZ-F-A and lacZ-R-A to amplify 1,023 bp of nucleotide sequence specific for the lacZ gene of Escherichia coli; and primers lacA-F2-A2 and lacA-R2-A2 to amplify 325 bp of nucleotide sequence specific for the lacA gene of Escherichia coli. As a result, 3 nucleotide fragments were generated when 3 samples DNA from Escherichia coli were used as template. On the other hand, 1,023- and 605-bp products were obtained when DNA of Shigella sonnei was used, and a 605-bp product was obtained when DNA of Shigella flexneri was used. The specificity of the technique was confirmed by comparing it with the conventional culture test; the consistency rate of both tests was 0.749. These results suggest that the technique described in the present study will be useful for distinguishing Escherichia coli from Shigella species with accuracy and specificity. Keywords Shigella
Escherichia coli Polymerase chain reaction
Communicated by Erko Stackebrandt. Y. Yamazaki and A. Fukasawa equally contributed to this work. Y. Yamazaki (&) A. Fukasawa Ibaraki prefectural institute of public health, 993-2, Kasarhara-cho, Mito, Ibaraki 310-0852, Japan e-mail: [email protected]
Introduction To survey the sanitation levels and the microbiological pollution in food and water, detection of indicator bacteria is of primary importance. Coliform bacteria and Escherichia coli are used as indicators of faecal pollution. E. coli is usually preferred as an indicator because it is specific and the most accurate in reflecting matter of faecal origin (Rompre et al. 2002). The Colilert test is a well-established culture method developed by IDEXX Laboratories Inc. (Portland, Maine) and is usually used to monitor the levels of or detect coliform bacteria and E. coli in wastewater and/or drinking water, including well water, because this test has high specificity and sensitivity, which is as good as or even better than the standard multiple-tube lactose fermentation method or membrane filter method (Edberg et al. 1989; Edberg et al. 1990). This test is based on a colorimetric reaction that produces b-galactosidase with the substrate o-nitrophenyl-p-galactopyranoside and indicates the presence of coliform bacteria and that produces b-Dglucuronidase with enzymatic transformation of the fluorogenic substrate 4-methylumbelliferyl-b-glucuronide and indicates the presence of E. coli (Edberg and Kontnick 1986; Edberg et al. 1989). However, it was found that th
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