Evaluation of DNA Extraction Methods for Culture-Independent Real-Time PCR-Based Detection of Listeria monocytogenes in Cheese

Abstract

Listeria monocytogenes, the causative agent of listeriosis, is a foodborne pathogen with significant public health and economic impacts. The control of pathogen presence in food requires rapid and sensitive methods. Real-time PCR is considered to be a fast and accurate tool for the detection of foodborne pathogens. A crucial step for the success of a culture-independent PCR-based detection is the template DNA extraction. Two open-formula extraction procedures and five kits were used to extract DNA prior to detection of L. monocytogenes in cheese. The extraction procedures were evaluated in quantitative PCR analyses of the total extracted bacterial DNA from cheese samples artificially contaminated with L. monocytogenes. The results of the study suggested that all seven extraction procedures can be used to obtain intact and amplifiable bacterial DNA, although with different L. monocytogenes detection limits in four types of cheese. PowerFood Microbial DNA Isolation Kit and DNeasy Mericon Food Kit performed the best with detection limits of 2.1 × 102 to 4.7 × 102 CFU/g in all analysed cheese samples. No differences in PCR detection limits of low numbers were found when different L. monocytogenes serotypes (1/2a, 1/2b, 1/2c and 4b) were used, despite the relatively lower DNA extraction efficiency observed in L. monocytogenes serotype 1/2c strain. Six out of seven evaluated methods demonstrated quantitative response with linear calibration lines and quantification limits of 4.6 × 102 to 9.3 × 103 CFU/g. The results demonstrated that culture-independent sample preparation and total DNA extraction, followed by target-specific DNA amplification have a potential to direct determination of low L. monocytogenes numbers in cheese within hours.