Comparison of the partial sequence of mdh genes and designing of PCR primers for the detection of Escherichia coli and Shigella spp.

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The presence of Escherichia coli has traditionally been considered an indicator microorganism of fecal contamination in food and in environmental water, and few of them are pathogenic to cause serious disease and food poisoning. So E. coli is therefore a widely used measure of sanitary quality. The common method for detecting and quantifying E. coli requires a minimum of 5 to 7 days to obtain results after initiation of sample analysis. Incorporated MUG to culture broth (i.e MUG-LST) were developed, but it could yield false-positive and false-negative results. The time and accuracy limitation suggested the need for more rapid methods for detecting E. coli. The first purpose of the study is to design PCR primers for the specific detection of the mdh gene of E. coli strains. Positively amplified DNA bands generated for all tested E. coli and Shigella spp. cells collected in our laboratory, and non-E. coli strains would not produce the specific PCR product. The detection sensitivity of Emdh1/ Emdh2 primers (25 pmole each) was 100 CFU per assay. In detection of E. coli cells in milk samples, tap water, underground water and lake water, the detection sensitivity would be 100 CFU if a pre-enrichment step using MacConkey broth was performed prior to the PCR. Owing the DNA relationship of E. coli and Shigella are homologous (about 70%~ 100%) in molecular level was reported. So the second work were sequencing Emdh1/ Emdh2 PCR products and structural gene in mdh gene of Shigella spp. for understanding the similarity. The results showed that the homology of E. coli and Shigella spp. was up to 98%. On the third part of the work, we have tried to develop PCR methods for the differentiation of viable and non-viable cells. By short term culture of the target cells followed by suitable dilution and PCR assay, non-viable cells could be differentiated from the viable cells since non-viable cells could not grow and its DNA would not give any PCR product due to dilution out of the cells. The detection sensitivity of viable cells were 100 CFU per assay, but the detection sensitivity of non-viable cells treated with autoclave sterilized, boiled-killing, Presterization and 70% EtOH were 102 CFU per assay. If a 8 hours pre-enrichment step was performed prior to PCR, could not generate target PCR products even the original non-viable cells number were 105 cells. Finally, mdh gene specific PCR primers Emdh1/ Emdh2 and MDH31/ MDH2 were combined into a multiplex PCR system and used for the simultaneous detection of E. coli and Salmonella typhimurium. On detection of E. coli and S. typhimurium cells in whole milk and drinking water, if a 8 hours pre-enrichment step was performed prior to PCR, the detection sensitivity for target cells in samples would be reach to 100/ 100 CFU per assay. When two target strains were different numbers mixed to the food samples, however, it was found that the ratio of original cell numbers should be within 102 so that the target strains with lower cell numbers could be detectable.