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

Abstract

Escherichia coli has been the appropriate focus for monitoring of potential enteric pathogens in water and foods. Although several methods have been used for the detection or enumeration of E. coli cells in water and foods, the time and accuracy limitations of these methods suggest the need of a rapid and specific method. By comparison of the gene sequences coding for malic acid dehydrogenase ( mdh) of E. coli and non- E. coli strains, two oligonucleotides were designed and their possible use as E. coli-specific PCR primers was tested. All of the 110 E. coli strains tested, including non-pathogenic and various pathogenic strains, generated the expected PCR products with M w equal to 392 bp. On the other hand, only 97 of these 110 E. coli strains were detectable using the BAM gas production method. With the exception of Shigella strains, non- E. coli strains, including strains of the family of Enterobacteriaceae, did not generate any false positive PCR results. When this PCR system was used for the monitoring of E. coli cells inoculated into water and milk samples, as low as 10 0 cfu per 100 ml of water or per ml of milk sample could be detected if an 8 h preculture step was performed prior to the PCR. Including the preculture step, the whole PCR detection process may be completed within 12 h.