Background: Vibrio cholerae and Escherichia coli were main causative agent foodborne diseases, especially in many developing countries, such as Indonesia. Thereby, rapid detection of these pathogenic bacteria is necessary to quickly detect infection that occurred so it can be treated immediately. In this case, multiplex PCR allows multiple genes amplification in one reaction thereby enable to perform rapid detection of these pathogenic bacteria. The objective of this study is to develop rapid molecular detection of V. Cholerae and E. coli and analyze the sensitivity and specificity of this assay.Result: In this study, we used various virulence genes in each pathogenic bacteria as marker to develop rapid molecular detection. Based on this research, optimum results of V. cholerae and E. coli rapid detection were obtained with a primer concentration of 16 µM for ctxA and ompU, 30 µM for ace, and 50 µM for zot, and toxR; 2 µM for elt and 5 µM for stx, respectively. Finally, based on the method standardization by ISO/TS 20836 these assays had 0% false positive, 0% false negative, 100% specificity, and 100% sensitivity; 0% false positive, 4% false negative, 100% specificity, and 96% sensitivity for V. cholerae and E. coli respectively. Conclusion: The optimized method was qualified to be used as a detection method for V. cholerae and E. coli detection according to ISO/TS 20836 (2017) and EHEC and ETEC contamination in drinking water samples.

Vibrio cholerae and pathogenic Escherichia coli were considered as main causative agent foodborne diseases especially in many developing countries, such as Indonesia. Thereby, rapid detection of these pathogenic bacteria is necessary to treat food-borne related diseases causing by these bacteria. In this case, multiplex PCR allows multiple genes amplification in one reaction thereby enable to perform rapid detection of these pathogenic bacteria. The objective of this study is to optimize uniplex and multiples PCR of V. cholerae and pathogenic E. coli detection and determine the sensitivity and specificity of this assays. We used various virulence genes for each pathogenic bacterium as markers for uniplex and multiplex PCR detection. Based on this research, the optimum results of V. cholerae and pathogenic E. coli were obtained with a primer concentration of 16 µM for ctxA and ompU, 30 µM for ace, and 50 µM for zot, and toxR; 2 µM for elt and 5 µM for stx, respectively. Finally, based on the standardization method by ISO/TS 20836 these assays had 0% false positive, 0% false negative, 100% specificity, and 100% sensitivity; 0% false positive, 4% false negative, 100% specificity, and 96% sensitivity for V. cholerae and pathogenic E. coli respectively. The optimized method was qualified to be used as a molecular detection for V. cholerae as well as EHEC and ETEC detection according to ISO/TS 20836 (2017)  from drinking water samples.