Listeria Monocytogenes In Food Samples Research Articles

LAMP based electrochemical sensor for extraction-free detection of Listeria monocytogenes in food samples

Listeria monocytogenes (LMO) represents one of the most serious hazards for food health due to the high mortality rate and its ability to survive at limiting conditions. In this study, an electrochemical sensor for the detection of LMO in food samples using loop mediated isothermal amplification (LAMP) technique is presented. The extraction step for the release of the nucleic acids of the sample was simplified using three different alternatives (QuickExtract buffer lysis, heating method and an extraction-free option). The three protocols were tested with the LAMP reaction in seven different food matrices (Dairy milk, fresh cheese, ham, hummus, lettuce, milk powder and pâté) and were able to detect 1 cfu/25 g as stipulated by the food safety regulations. It was proved that all three alternatives were compatible with the electrochemical detection using methylene blue redox-active molecule in dairy milk samples. The final validation of the sensor was carried out with the extraction-free option in nine different food matrices, which were able to detect 1 cfu/25 g of food sample in all cases. This validation demonstrates the feasibility of the sensor for its application in the food industry as a point-of-test solution.

Quartz crystal microbalance–based aptasensor integrated with magnetic pre-concentration system for detection of Listeria monocytogenes in food samples

A fast and accurate identification of Listeria monocytogenes. A new quartz crystal microbalance (QCM) aptasensor was designed for the specific and rapid detection of L. monocytogenes. Before detection of the target bacterium from samples in the QCM aptasensor, a magnetic pre-enrichment system was used to eliminate any contaminant in the samples. The prepared magnetic system was characterized using ATR-FTIR, SEM, VSM, BET, and analytical methods. The saturation magnetization values of the Fe3O4, Fe3O4@PDA, and Fe3O4@PDA@DAPEG particles were 57.2, 40.8, and 36.4 emu/g, respectively. The same aptamer was also immobilized on the QCM crystal integrated into QCM flow cell and utilized to quantitatively detect L. monocytogenes cells from the samples. It was found that a specific aptamer-magnetic pre-concentration system efficiently captured L. monocytogenes cells in a short time (approximately 10 min). The Fe3O4@PDA@DA-PEG-Apt particles provided selective isolation of L. monocytogenes from the bacteria-spiked media up to 91.8%. The immobilized aptamer content of the magnetic particles was 5834 µg/g using 500 ng Apt/mL. The QCM aptasensor showed a very high range of analytical performance to the target bacterium from 1.0 × 102 and 1.0 × 107 CFU/mL. The limit of detection (LOD) and limit of quantitation (LOQ) were 148 and 448 CFU/mL, respectively, from the feeding of the QCM aptasensor flow cell with the eluent of the magnetic pre-concentration system. The reproducibility of the aptasensor was more than 95%. The aptasensor was very specific to L. monocytogenes compared to the other Listeria species (i.e., L. ivanovii, L. innocua, and L. seeligeri) or other tested bacteria such as Staphylococcus aureus, Escherichia coli, and Bacillus subtilis. The QCM aptasensor was regenerated with NaOH solution, and the system was reused many times.Graphical

Development and Application of Four Foodborne Pathogens by TaqMan Multiplex Real-Time PCR.

A TaqMan multiplex real-time PCR (mRT-PCR) was developed to detect simultaneously Salmonella spp., Escherichia coli O157, Staphylococcus aureus, and Listeria monocytogenes in food samples. The method involves four sets of primers and probes tailored to the unique DNA sequences found in the invA, nuc, rfbE, and hly genes of each pathogen. The generated standard curves, correlating gene copy numbers with Ct values, demonstrated high accuracy (R2 > 0.99) and efficiency (92%-104%). Meanwhile, the limit of detection was 100 CFU/mL for the four target bacteria in artificially contaminated food samples after 6-8 h of enrichment. The assay's effectiveness was further verified by testing 80 naturally contaminated food samples, showing results largely in agreement with traditional culture methods. Overall, this newly developed TaqMan mRT-PCR, inclusive of a pre-enrichment step, proves to be a dependable and effective tool for detecting single or multiple pathogens in diverse food items, offering significant potential for in vitro diagnostics.

Development of Optical Label-Free Biosensor Method in Detection of Listeria monocytogenes from Food.

The present work describes an alternative method for detecting and identifying Listeria monocytogenes in food samples by developing a nanophotonic biosensor containing bioreceptors and optical transducers. The development of photonic sensors for the detection of pathogens in the food industry involves the implementation of procedures for selecting probes against the antigens of interest and the functionalization of the sensor surfaces on which the said bioreceptors are located. As a previous step to functionalizing the biosensor, an immobilization control of these antibodies on silicon nitride surfaces was carried out to check the effectiveness of in plane immobilization. On the one hand, it was observed that a Listeria monocytogenes-specific polyclonal antibody has a greater binding capacity to the antigen at a wide range of concentrations. A Listeria monocytogenes monoclonal antibody is more specific and has a greater binding capacity only at low concentrations. An assay for evaluating selected antibodies against particular antigens of Listeria monocytogenes bacteria was designed to determine the binding specificity of each probe using the indirect ELISA detection technique. In addition, a validation method was established against the reference method for many replicates belonging to different batches of meat-detectable samples, with a medium and pre-enrichment time that allowed optimal recovery of the target microorganism. Moreover, no cross-reactivity with other nontarget bacteria was observed. Thus, this system is a simple, highly sensitive, and accurate platform for L. monocytogenes detection.

Optimization of a Viability PCR Method for the Detection of Listeria monocytogenes in Food Samples.

Rapid detection of Listeria and other microbial pathogens in food is an essential part of quality control and it is critical for ensuring the safety of consumers. Culture-based methods for detecting foodborne pathogens are time-consuming, laborious and cannot detect viable but non-culturable microorganism, whereas viability PCR methodology provides quick results; it is able to detect viable but non-culturable cells, and allows for easier handling of large amount of samples. Although the most critical point to use viability PCR technique is achieving the complete exclusion of dead cell amplification signals, many improvements are being introduced to overcome this. In the present work, the yield of dead cell DNA neutralization was enhanced by incorporating two new sample treatment strategies: tube change combined with a double light treatment. This procedure was successfully tested using artificially contaminated food samples, showing improved neutralization of dead cell DNA.

A new method for rapid detection of Listeria monocytogenes in food

Listeria monocytogenes represents serious danger for human health. Thus detection of this pathogen in food, which represents its main means of entry into the organism, is a topic of special importance. The original classic methods for the determination of Listeria monocytogenes are in general laborious and time-consuming procedures. In order to address this issue we developed a new rapid method for specific detection of Listeria monocytogenes in food samples. The method consists of three steps: i) enrichment of food microflora (16 h), ii) selective isolation of Listeria sp. exploiting immunomagnetic separation (2–3 h) followed by iii) precise identification of Listeria sp. and Listeria monocytogenes using duplex PCR. PCR primers specific to part of 16S rRNA were used in order to identify the members of Listeria genus. The specific identification of Listeria monocytogenes was accomplished exploiting a pair of primers specific to gene encoding invasion-associated protein – iap (4–5 h). Amplification products, 1003 bp and 593 bp respectively, were separated by electrophoresis and visualized by UV detection. The optimized IMS-PCR method was used to test the presence of Listeria sp. and Listeria monocytogenes in food samples (ground meat, low-fat milk and cheese [olomoucké tvarůžky]). A comparison of the efficiency of the bacteria enrichment step by IMS and centrifugation was also performed. The analysis time including enrichment is less than 24 h. The detection limit for Listeria monocytogenes was found between 101–102 cfu per 25 g of food sample.

Actuation of chitosan-aptamer nanobrush borders for pathogen sensing.

We demonstrate a sensing mechanism for rapid detection of Listeria monocytogenes in food samples using the actuation of chitosan-aptamer nanobrush borders. The bio-inspired soft material and sensing strategy mimic natural symbiotic systems, where low levels of bacteria are selectively captured from complex matrices. To engineer this biomimetic system, we first develop reduced graphene oxide/nanoplatinum (rGO-nPt) electrodes, and characterize the fundamental electrochemical behavior in the presence and absence of chitosan nanobrushes during actuation (pH-stimulated osmotic swelling). We then characterize the electrochemical behavior of the nanobrush when receptors (antibodies or DNA aptamers) are conjugated to the surface. Finally, we test various techniques to determine the most efficient capture strategy based on nanobrush actuation, and then apply the biosensors in a food product. Maximum cell capture occurs when aptamers conjugated to the nanobrush bind cells in the extended conformation (pH < 6), followed by impedance measurement in the collapsed nanobrush conformation (pH > 6). The aptamer-nanobrush hybrid material was more efficient than the antibody-nanobrush material, which was likely due to the relatively high adsorption capacity for aptamers. The biomimetic material was used to develop a rapid test (17 min) for selectively detecting L. monocytogenes at concentrations ranging from 9 to 107 CFU mL-1 with no pre-concentration, and in the presence of other Gram-positive cells (Listeria innocua and Staphylococcus aureus). Use of this bio-inspired material is among the most efficient for L. monocytogenes sensing to date, and does not require sample pretreatment, making nanobrush borders a promising new material for rapid pathogen detection in food.

Establishment and Application of a Visual DNA Microarray for the Detection of Food-borne Pathogens.

The accurate detection and identification of food-borne pathogenic microorganisms is critical for food safety nowadays. In the present work, a visual DNA microarray was established and applied to detect pathogens commonly found in food, including Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in food samples. Multiplex PCR (mPCR) was employed to simultaneously amplify specific gene fragments, fimY for Salmonella, ipaH for Shigella, iap for L. monocytogenes and ECs2841 for E. coli O157:H7, respectively. Biotinylated PCR amplicons annealed to the microarray probes were then reacted with a streptavidin-alkaline phosphatase conjugate and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP); the positive results were easily visualized as blue dots formatted on the microarray surface. The performance of a DNA microarray was tested against 14 representative collection strains and mock-contamination food samples. The combination of mPCR and a visual micro-plate chip specifically and sensitively detected Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in standard strains and food matrices with a sensitivity of ∼10(2) CFU/mL of bacterial culture. Thus, the developed method is advantageous because of its high throughput, cost-effectiveness and ease of use.

Multi-Province Listeriosis Outbreak Linked to Contaminated Deli Meat Consumed Primarily in Institutional Settings, Canada, 2008.

A multi-province outbreak of listeriosis occurred in Canada from June to November 2008. Fifty-seven persons were infected with 1 of 3 similar outbreak strains defined by pulsed-field gel electrophoresis, and 24 (42%) individuals died. Forty-one (72%) of 57 individuals were residents of long-term care facilities or hospital inpatients during their exposure period. Descriptive epidemiology, product traceback, and detection of the outbreak strains of Listeria monocytogenes in food samples and the plant environment confirmed delicatessen meat manufactured by one establishment and purchased primarily by institutions was the source of the outbreak. The food safety investigation identified a plant environment conducive to the introduction and proliferation of L. monocytogenes and persistently contaminated with Listeria spp. This outbreak demonstrated the need for improved listeriosis surveillance, strict control of L. monocytogenes in establishments producing ready-to-eat foods, and advice to vulnerable populations and institutions serving these populations regarding which high-risk foods to avoid.

Detection of Listeria monocytogenes in food samples in Avezzano, Sulmona and Castel di Sangro (province of L'Aquila, Abruzzo, Italy) between 2000-2009.

The retrospective study of the results of the analysed samples is a fundamental tool for the identification of major risk related to food and for planning future monitoring activities. The evaluation of the quality of data collected may also allow for estimating the effectiveness of the controls so to improve their efficacy. In this article, the authors evaluated the results of tests for the detection of Listeria monocytogenes performed by the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' (IZSAM) on food samples collected during the years 2000-2009 in the territory of Avezzano, Sulmona and Castel di Sangro (province of L'Aquila, Abruzzo, Italy). The comparison of the data examined with those from studies conducted in Italy and in other countries shows that the categories with higher percentages of positivity for Listeria monocytogenes are meat and fish products. Data collected do not indicate cheese as a vehicle of contamination in the sampled areas, in contrast to what reported in the national and international literature. It would therefore be necessary to promote an ad hoc sampling in the areas covered by this study to verify this aspect in more depth.

Taq Man Real Time PCR detection of Listeria monocytogenes: A study of enrichment incubation time affecting sensitivity in experimental dry fermented sausages

Listeria monocytogenes is a facultative intracellular Gram positive, catalase positive bacterium, ubiquitous in nature and capable of causing listeriosis in humans and animals. Conventional microbiological techniques and modern molecular approaches are currently used for the isolation and detection of Listeria monocytogenes in food samples. The aim of this study was to investigate the influence of enrichment incubation time on the sensitivity of Taq Man Real Time PCR method. For that purpose, dry fermented sausages were artificially inoculated with serial dilutions of L. monocytogenes ATCC 19111. The obtained results indicated that incubation time is an important factor affecting the sensitivity of Real-Time PCR detection. The best results were obtained after 24 h of pre-enrichment, with primers and probe complementary to the listeriolysin (hlyA) gene, when it was possible to detect less than 10 CFU/g of L. monocytogenes.

Inhibitory effects of Listeria selective enrichment media on growth characteristics of L. ivanovii

Various standard methods by different food regulatory bodies have been proposed different selective enrichment to grow and isolate Listeria monocytogenes in food samples. Different studies have demonstrated a variety of enrichment strategies to effectively improve the isolation rate of Listeria. However, very limited research was dedicated to understand the kinetics of Listeria spp. growth under imposed sublethal stresses by common selective agents used in those conventional enrichment media. A comprehensive list of primary and secondary enrichment media (i.e., UVM, LEB, SEB, HFB, PB, DFB, BLEB, MOPS-BLEB and FB) were used to study L. ivanovii multiplication at 37oC incubation for 24 hr. The medium concentrations were varied at four levels; 25%, 50%, 100% and 200% to accentuate the inhibitory effect of indigenous selective agents. The net normalized growth rate and the total cell count were utilized to evaluate the lethality of each selective media and survivability of L. ivanovii. The toxicity of the media to L. ivanovii was escalated significantly and produced negative impact on growth as the strength of the enrichment medium concentration increased (25% to 200%). The negative growth effect was more pronounced for all secondary enrichment media used. Only did LEB and SEB return positive net growth rate and produce the least negative impact on the cell multiplicity.

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

We developed a novel filtration-based method that can eliminate dead or severally damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences ( p < 0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear ( R 2 > 0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.

Antibody Immobilization on Waveguides Using aFlow–Through System Shows Improved Listeria monocytogenesDetection in an Automated Fiber Optic Biosensor: RAPTORTM

Recent outbreaks of food borne illnesses continue to support the need for rapidand sensitive methods for detection of foodborne pathogens. A method for detecting Listeriamonocytogenes in food samples was developed using an automated fiber-optic-basedimmunosensor, RAPTORTM. Detection of L. monocytogenes in phosphate buffered saline(PBS) was performed to evaluate both static and flow through antibody immobilizationmethods for capture antibodies in a sandwich assay. Subsequent detection in frankfurtersamples was conducted using a flow through immobilization system. A two stage blockingusing biotinylated bovine serum albumin (b-BSA) and BSA was effectively employed toreduce the non-specific binding. The sandwich assay using static or flow through mode ofantibody immobilization could detect 1

Detection of Listeria monocytogenes from Food Samples by PCR after IMS-Plating

For rapid, accurate and sensitive detection of Listeria monocytogenes in food samples, colonies developed on the selective agar (Oxford agar) after immunomagnetic separation (IMS) were subjected to polymerase chain reaction (PCR) assay with the prf A1-2 primer pair. The proposed assay system was shown experimentally to be capable of specifically detecting the bacteria from food samples contaminated at more than 10(2) cfu/g. However, the enrichment culture after a short period of 16 h with the appropriate selective broth was needed before IMS-plating, because the bacterial contents in most actual food were as low as less than 10(2) cfu/g. However, even if the enrichment cultivation was employed before IMS, L. monocytogenes was detected within 3 days.

Impact of enrichment medium on PCR-based detection of Listeria monocytogenes in food

 The detection of Listeria monocytogenes in food samples using enrichment and PCR is described. The objectives were to determine whether the type of enrichment medium has the influence on the results obtained with PCR and to evaluate proposed method by analysing naturally contaminated food samples. Comparative evaluation of different enrichment media for bacteria of the genus Listeria (half Fraser - HF, buffered peptone water - BPW, triptic soy yeast extract broth -  TSBYE, universal preenrichment broth - UPB) showed that only the UPB enabled detection of L. monocytogenes in artificially contaminated food samples after 24h of incubation. The PCR-based method gave equal results as standard cultural method by analysis of naturally contaminated food samples. Described PCR-based procedure is comparable to some other PCR-based methods of L. monocytogenes detection in foods and shows promise as a rapid, routine method which requires 30-72 h, whereas cultural methods require 96-120 h.

Use of specific oligonucleotides for direct enumeration of Listeria monocytogenes in food samples by colony hybridization and rapid detection by PCR

Two 18-mer oligonucleotides derived from the sequence of hly, the gene coding for listeriolysin O, were shown to be specific for Listeria monocytogenes in the genus Listeria in colony hybridization tests. The oligonucleotides did not hybridize with any of the bacterial species found in food and co-isolated with Listeria on selective media. They were used in colony hybridization tests for enumeration of L. monocytogenes present in food samples after direct plating on selective media plates. In addition, two 24-mer oligonucleotides, each including the sequence of one of the 18-mers, were successfully used for the PCR-based detection of L. monocytogenes bacilli present in food samples after a 48-h enrichment period. Using this technique, as little as 10 2 bacteria per ml of enrichment broth can be detected.

A rapid polymerase chain reaction (PCR)-based assay for the identification of Listeria monocytogenes in food samples

A rapid and simple assay has been developed which allows specific detection of Listeria monocytogenes within 3.5 h in cultures prepared from suspect food samples and propagated 48 h in selective medium. The assay is based on PCR technology, and uses a specific primer set derived from sequences located down-stream of the hlyA gene. The specificity of the primer set was confirmed by testing 115 L. monocytogenes, 14 L. innocua, 5 L. seeligeri and 4 L. ivanovii isolates. The assay was compared to standard microbiological tests and gave identical results for 83 food samples, including 32 positives. These field trials indicate that the assay developed provides an alternative detection system for L. monocytogenes in foods, which can be used by the food industry.