PCR primers designed from malic acid dehydrogenase gene and their use for detection of Escherichia coli in water and milk samples

Escherichia coli has been the appropriate focus for monitoring of potential enteric pathogens in water and foods. Although several methods have been u sed for the detection or enumeration of E. coli cells in water and foods, t he time and accuracy limitations of these methods suggest the need of a rap id and specific method. By comparison; of the gene sequences coding for mal ic acid dehydrogenase (mdh) of E. coli and non-E. coli strains, two oligonu cleotides were designed and their possible use as E. coli-specific PCR prim ers was tested. All of the 110 E. coli strains tested, including non-pathog enic and various pathogenic strains, generated the expected PCR products wi th M-w equal to 392 bp. On the other hand, only 97 of these 110 E. roll str ains were detectable using the BAM gas production method. With the exceptio n of Shigella strains, non-E. coli strains, including strains of the family of Enterobacteriaceae, did not generate any false positive PCR results. Wh en this PCR system was used for the monitoring of E. coli cells inoculated into water and milk samples. as low; as 10 degrees cfu per 100 ml of water or per ml of milk sample could be detected if an 8 h preculture step was pe rformed prior to the PCR. Including the preculture step. the whole PCR dete ction process may be completed within 12 h. (C) 2001 Elsevier Science B.V. All rights reserved.