A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 ؋ 10 2 CFU/cm 2 .In the food industry, Listeria monocytogenes represents an important health risk. The consumption of food products contaminated with this gram-positive bacterium can cause listeriosis, a serious disease with a 30% mortality rate. Raw material can contain L. monocytogenes, but contamination of food products can also occur during processing. The rapid methods currently available for identifying L. monocytogenes are limited by a threshold of approximately 10 5 CFU/ml and consequently require enrichment procedures (22). Rapid quantification of L. monocytogenes is important for many reasons, such as in the Hazard Analysis and Critical Control Point method program to verify critical limits and monitor contamination levels and for quantitative risk assessment. L. monocytogenes, like several food-borne pathogens, can attach to the surfaces in contact with the products, leading to the formation of biofilms (12,14). Adherent bacteria can acquire new physiological properties compared with planktonic cells conferring resistance to disinfectants or sanitizers, leading to hygiene problems, particularly for pathogens that can participate in the recontamination of products (7,19). Rapid enumeration of L. monocytogenes in biofilms could thus be very useful in the identification of sources of food contamination. DNA-based methods, such as PCR, have been considerably developed to detect food-borne pathogens like L. monocytogenes (1,6,8,21). Real-time PCR has previously been proposed to detect and quantify L. monocytogenes in food products like milk, cheese, and cabbage (10, 13, 18). However, to our knowledge, no study has been reported concerning real-time PCR for the detection and quantification of L. monocytogenes in biofilms.The aim of this work was to develop a quantitative method to evaluate the population of L. monocytogenes in artificially made biofilms. The real-time PCR can be used to estimate the number of copies of a target gene in a sample and is reported to be more sensitive than conventional qualitative PCR. Realtime PCR is based on the detection and quantification of a fluorescent reporter, whose emission is directly proportional to the quantity of amplicons generated during the PCR. The fluorescent reporter used in this study was SYBR Green I, a nonspecific double-stranded DNA-binding dye. SYBR Green I has the advantage of not requiring the design of specific probes, and its binding is not affected by potential mutations of the target gene. The specificity and sensitivity of the primers used were determined, and then the effectiveness of four methods of extracting DNA from L. monocytogenes growing in biofilms was evaluated. Finally, the detection and quantification of L. monocytogen...
