COMPARISON, VALIDATION, AND OPTIMIZATION OF INTERNAL GENOMIC DNA EXTRACTION PROTOCOL FOR CAMPYLOBACTER SPECIES

Abstract

Campylobacter remains the leading cause responsible of human gastroenteritis worldwide. The current study aimed to compare the internal DNA extraction protocol with two commercially available kits, using C. jejuni (ATCC^® 29428^TM) and C. coli (ATCC^®43478^TM) and to validate and optimize the internal protocol through artificial contamination and confirmation of Campylobacter spp. from broiler chickens, turkeys, and beef meats samples. The extraction processes were carried out following the internal protocols steps and the manufacturer's instructions of PureLink^TM Genomic DNA Mini Kit and Wizard^® Genomic DNA Purification Kit, respectively. The agarose gel electrophoresis system was used to control the DNA quality. After that, forty Campylobacter spp. isolates were confirmed, and finally, an artificial contamination of the aforementioned reference strains at three different concentrations was performed with sterilized minced meats. This is the first work comparing the PureLink^TM Genomic DNA Mini Kit and Wizard^® Genomic DNA Purification Kit with internal genomic DNA extraction protocol for Campylobacter spp. The results indicated that the internal protocol provided similar efficiency to the two kits. All confirmed isolates were successfully amplified, in which28 isolates were C. coli and 12 were C. jejuni, as revealed by biochemical tests. A positive amplification was also observed in the three contaminated food matrices, after enrichment, at all examined doses. Except some reactions that were negative at 1 CFU/mL of C. jejuni and C. coli. This was explained by the detection limits of both internal protocol and qPCR. Based on our findings, three crucial steps in determining the extraction of DNA quality of this protocol were amended. Hence, the study highlighted the importance of validating simpler, cheaper and faster DNA extraction protocol, for each laboratory, as part of future risk assessment, control and monitoring programs of Campylobacter frequency required in molecular studies.