Real-Time PCR Method Combined with a Matrix Lysis Procedure for the Quantification of Listeria monocytogenes in Meat Products

Abstract

In this study a real-time PCR method has been developed for the specific quantification of the foodborne pathogen Listeria monocytogenes on meat products through the gene hlyA. The PCR was combined with a matrix lysis that allowed the obtaining of the microorganisms without sample dilution and the elimination the PCR inhibitors from dry-cured ham. The qPCR method calibration curve had an efficiency of 100.4%, limits of detection and quantification were 30.1 ± 6.2 CFU/g which is under the legal limit of L. monocytogenes in ready-to-eat products, and an analytical variability <0.25 log hlyA gene copies/reaction. The analysis was performed simultaneously with the reference method ISO 11290-2. The comparison of the qPCR-matrix lysis results with the reference method showed an excellent correspondence, with a relative accuracy between 95.83–105.20%. Finally, the method was applied to commercial derived meat samples and the pathogen was quantified in one of the commercial samples assayed in 69.1 ± 13.9 CFU/g while the reference method did not quantify it. The optimized qPCR showed higher precision and sensitivity than the reference method at low concentrations of the microorganism in a shorter time. Therefore, qPCR-matrix lysis shows a potential application in the meat industry for L. monocytogenes routine control.

Improving the Efficiency of Viability-qPCR with Lactic Acid Enhancer for the Selective Detection of Live Pathogens in Foods

“…For UHT milk, the standard plating methods require different enrichment media plus long detection/confirmation times (2 days) to target aerobic or anaerobic viable contaminant bacteria. When it comes to analyzing food samples using qPCR, the complexity of food matrices poses several challenges: the presence of PCR inhibitors, variability in food composition, texture, and structure, the presence of enzymes that degrade DNA, and so on [46][47][48][49][50]. In our experiments, the lowest detection limit was log 2 CFU/mL for viable cells or mixtures with log 2 CFU/mL alive and log 2 CFU/mL heat-killed bacteria.…”

Identification by High-Throughput Real-Time PCR of 30 Major Circulating Listeria monocytogenes Clonal Complexes in Europe

“…Real-time PCR technology offers the possibility to rapidly detect L. monocytogenes with higher specificity, sensitivity, and reliability than conventional PCR using agarose gel-based detection ( 46 ). A plethora of real-time PCR assays has been developed for detecting and quantifying L. monocytogenes in various food matrixes ( 46 – 48 ) and in water and environmental surfaces ( 49 ). These methods enable the identification of species or the five major molecular serogroups ( 50 , 51 ).…”

Detection of Listeria monocytogenes in Food Using the Proofman-LMTIA Assay

“…Biosensor-based techniques are constrained by higher cost regarding the short lifespan of electronic components, which restrict their application. Molecular biology techniques utilizing DNA, such as polymerase chain reaction (PCR) [ 13 ], loop-mediated isothermal amplification (LAMP) [ 14 ], droplet digital PCR [ 15 ], and real-time PCR [ 16 , 17 ], have proven to be highly effective in detecting Listeria monocytogenes in food. However, these methods often require expensive instruments and complex operations, resulting in long detection times.…”