Sequencing of the mdh genes of Shigella spp., E. coli, detection of enterotoxin gene and cytotoxicities for Salmonella spp. and the Simultaneous detection of E. coli O157:H7 as well as Salmonellae in foods using an Immunomagnetic separation-multiplex PCR

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Escherichia coli is an index orgainism for fecal contamination of foods and environments. Infection by pathogenic E. coli strains may cause human diarrhea disease or food poisoning case. On the food safety viewpoint, rapid method for E. coli detection is thus important. On E. coli detection, both the PCR and DNA hybridization methods have been developed. However, differentiation between E. coli and Shigella spp. has always been difficult. Previously, we have developed malate dehydrogenase gene (mdh) based PCR primers for the specific detection of E. coli cells, and found that Shigella spp. could generate false positive results. In this report, we found that by DNA sequencing technique, the mdh gene sequences for Shigella spp. show a sequence homology higher than 98% as compared to those of the E. coli cells. According to the results from in vivo and in vitro tests, Salmonella enterotoxin has been found important to the virulence of Salmonella cells. However, on enterotoxin assay, only part of the serovar of Salmonella strains have been investigated for the presence of enterotoxin (stn) gene. We thus, in this study, designed stn specific primers and used them for the survey of the presence of stn gene in most of the Salmonella strains including 80 Salmonella strains with 23 serovarieties. Furthermore, the CHO cell cytotoxicities for these Salmonella strains were also investigated so that the relationship between stn gene and virulence for Salmonella strains may be elucidated. Results show that for these 80 Salmonella strains, 93.8% show positive PCR results. 6.2% of these strains, ie, strains of Salm. oranienburg(SO 24), Salm. richmond(SR 03), Salm. sandiego(SS 15), Salm. tennessee(ST 182), Salm. vejle(SV 10) show negative PCR results. These five strains, are negative in cytotoxicity test using CHO as target cells. In addition, frequencies for isolation of these strains from infective cause are very low. Since both E. coli O157 and Salmonella spp. are important for food-poisoning cases, we thus also designed a multiplex PCR system which allowed the simultaneous detection of E. coli O157 and Salmonella spp. in foods. On using this multiplex PCR system, it was found that if the target cell number ratio were higher than 102, both target cells could not be detected simultaneously, even a preculture step using GN broth was performed prior to PCR. However, when immunomagnetic separation (IMS) method using equimolar mixture of anti-Salmonella and ant-E.coli O157 immuno-beads was performed prior to PCR, both target cells could be detected even the ratios of these target cells were up 105. Also, as the IMS-PCR method was used for PCR inspection of food samples, food components and the naturally contaminated non-Salmonella microflora could not interfere with the detection.