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Gram-negative bacteria that cause food poisoning became an important focus of attention in both developed and developing countries. These bacteria include Salmonella spp and Escherichia coli. In the previous stage, the conditions and reaction formula for the prototype rapid kit detections of foodborne pathogens were obtained using the Real Time PCR method. This research is a stage of validating the conditions and reaction formulas found in the previous stage, to obtain a consistent and reproducible prototype product. Validation of the isolation stage of Salmonella spp (Salmonella typhi, Salmonella typhimurium, Salmonella enteritidis) and Escherichia coli bacteria were carried out by cultivating cultures at 250 rpm aeration conditions, and temperature 37oC. A total of three mL of cultures was centrifuged at 5000 rpm and yielded each cell weight of 0.25-0.6 grams. The results of the isolation of genomic DNA with Qiaprep were 200 microliters with the amount of DNA 10600-11700 nanograms. The concentrations were 23-58.5 ng/mL, and the purities were 1.809-2.084. Furthermore, the resulting genomic DNA is used as a template for the PCR process. The validation of the PCR stage of Salmonella spp using annealing temperature of 60oC and template concentration of 50 ng resulted in the Salmonella typhi fim-C gene amplicons, Salmonella typhimurium pef gene and Salmonella enteritidis prot6E gene measuring 95 bp, 139 bp and 170 bp, respectively. PCR validation of the E. coli DNA template at 60oC with the same concentration resulted in an amplicon measuring 121 bp. Based on the data obtained, it can be concluded that the validation of the isolation stage and confirmed PCR produced data that were consistent with previous studies and were reproducible. With the result that the development of a detection kit prototype can be continued to the validation stage with Real Time PCR and laboratory-scale trials for artificially contaminated food samples and food samples from products on the market. [ABSTRACT FROM AUTHOR]

The case of foodborne disease by pathogenic bacteria is an extraordinary event whose source and route of spread occur quickly. Bacillus cereus and Listeria monocytogenes are included in the gram-positive bacteria that cause food poisoning infections in humans. In previous studies, the conditions and reaction formulas for the prototype rapid kit detections of foodborne pathogens were obtained using the Real-Time PCR method. This research validates the conditions and reaction formulas in the prior stage to obtain a consistent and reproducible prototype product. Validation of the isolation stage of Bacillus cereus and Listeria monocytogenes bacteria was carried out by culturing bacterial cultures at aeration conditions of 250 rpm and a temperature of 37oC. A total of three mL of the culture was centrifuged at 5000 rpm and yielded 0.03 and 0.02 grams of B. cereus and L. monocytogenes cells, respectively. The results of the isolation of genomic DNA with Qiaprep were 200 µL with the amount of DNA 10600-11700 nanograms; the concentration was 23-58.5 ng/μL, and the purity was 1.809-2.084. Furthermore, the resulting it is used as a template for the PCR process. The validation of the PCR stage of B. cereus and L. monocytogenes at a temperature of 60oC and template concentration of 50-60 ng resulted in an amplicon of both bacteria. The cyt-k gene B. cereus and hly L. monocytogenes are measuring respectively 114 bp and 158 bp. Based on the data obtained, it can be concluded that the validation of the isolation and PCR stages has been successfully carried out by producing data consistent with previous studies. The development of a detection kit prototype is then revealed to the Real-Time PCR stage and laboratory-scale trials for artificially contaminated food samples and food samples from products on the market. [ABSTRACT FROM AUTHOR]