Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Abstract

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE QPCR SYBR Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.

Real-Time PCR Assay To Differentiate Listeriolysin S-Positive and -Negative Strains ofListeria monocytogenes

“…To address the extensive labor and time required for the detection and identification of L. monocytogenes by conventional means, a number of rapid and highly sophisticated realtime PCR-based technologies have been developed (4,44,48,50,51). More specifically, such technologies are attractive due to the fact that culture-based methods, which typically involve selective enrichment, culturing on selective media, and a battery of biochemical tests and serology, can take from 5 to 10 FIG.…”

Development, and complete evaluation, of a novel Most-Probable-Number (MPN) qPCR method for accurate and express quantification of Listeria monocytogenes in foodstuffs

“…This fact represents a main advantage over previously published procedures which take a minimum of 40 h only in the incubation step, [23,44,45] and may take even longer if the confirmation steps are performed by traditional microbiology or conventional PCR [22,[46][47][48]. Additionally, our method demonstrated suitability for application with two different detection chemistries, SYBR Green intercalating dye, which is a more economic approach, and with a hydrolysis probe, which offers additional specificity.…”

A Fast, Reliable, and Sensitive Method for Detection and Quantification ofListeria monocytogenesandEscherichia coliO157:H7 in Ready-to-Eat Fresh-Cut Products by MPN-qPCR

“…Genomic DNA was extracted from dilution tubes considered for MPN enumeration by comparing two methods: the DNeasy Blood and Tissue kit (Qiagen, Milano, IT) according to the manufacturer's instructions and the boiling method reported by de Oliveira et al [16]. …”

“…Although MPN method has the advantage of enabling detection of the target pathogen even when it is present in low numbers, it is laborious and requires several days for confirmation of results. Therefore, in the last years, a considerable number of detection methods using faster molecular tools, mainly based on PCR techniques, have been proposed [16, 17, 21–23]. Currently, several genes have been suggested as targets for the molecular detection of L. monocytogenes [16, 24, 25] and E. coli O157:H7 [26].…”

“…Therefore, in the last years, a considerable number of detection methods using faster molecular tools, mainly based on PCR techniques, have been proposed [16, 17, 21–23]. Currently, several genes have been suggested as targets for the molecular detection of L. monocytogenes [16, 24, 25] and E. coli O157:H7 [26]. The most recent advances in PCR methods are focused on the reduction of the limits of detection and quantification [27, 28], on the discrimination between dead and live cells [22] or the simultaneous detection of different foodborne pathogens [22, 27–29].…”