Comparison and Optimization of Genomic DNAs Extraction of Four Pathogenic Bacteria by Genomic DNA Extraction Kit for Foodborne Microbial Detection

Objective: This study describes preparing genomic DNA samples of foodborne pathogens which meet the requirements of national nucleic acid reference materials with independent intellectual property rights by using the domestic bacterial genomic DNA extraction kit. Methods: Four foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enteritidis and Staphylococcus aureus were used in this study. The imported OMEGA bacterial genomic DNA extraction kit and the domestic TIANGEN bacterial genomic DNA extraction kit were used to extract the genomic DNAs of the four pathogens. And the genomic DNA extraction methods of E. coli O157:H7, S. enteritidis and S. aureus with low concentration and purity extracted from domestic TIANGEN kit were optimized. Finally, the integrity and purity of the genomic DNAs of the four pathogens were detected by agarose gel electrophoresis, and the characteristic virulence genes of the four pathogens were verified by PCR amplification. Results: The genomic DNAs' mass concentrations of the four pathogens extracted by the domestic TIANGEN bacterial genomic DNA extraction kit were greater than or equal to 50 ng/μL, and the values of A260/A280 and A260/A230 were between 1.7-2.0. The bands of the genomic DNAs are clear, free of RNA and protein contamination, and the characteristic virulence genes of each strain are positive. Conclusion: Through the optimization of genomic DNA extraction method, four pathogen genomic samples conforming to the requirements of national nucleic acid standard materials were obtained, which laid a foundation for the subsequent preparation of large quantities of nucleic acid standard samples. © 2020, Editorial Office of Journal of CIFST. All right reserved.