Specific Detection Of Listeria Monocytogenes Research Articles

Aptamer and Ru(bpy)32+‐AuNPs‐based electrochemiluminescence biosensor for accurate detecting Listeria monocytogenes

AbstractAs a widespread foodborne pathogen, Listeria monocytogenes (LM) can potentially cause a large number of food safety incidents, and the mortality rate of humans infected with this pathogen is relatively high. Thus, establishment of a fast and simple inspection technology is imperative. Herein, a solid‐state electrochemiluminescence (ECL) biosensing switch system based on special ferrocene‐labeled molecular beacon aptamer (Fc‐MBA) has been developed for rapid detection of LM. The biosensor consisted of two main parts, an ECL substrate and an ECL intensity switch. ECL substrate was generated by modifying the complex of gold nanoparticle (AuNPs) and ruthenium (II) tris‐(bipyridine) onto Au electrode, and an MBA labeled by Fc that acted as an ECL intensity switch. Moreover, in the presence of LM, the stem‐loop structure of MBA could be opened and the ferrocene was kept away from ECL substrate, which was due to an obvious ECL intensity increment. The limit of detection was 5 CFU/mL, and satisfactory recoveries of 98.1–103.6% were observed. Thus, we propose a novel method for accurate and specific detection of LM.

Phage Display-Derived Monoclonal Antibodies Against Internalins A and B Allow Specific Detection of Listeria monocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, a highly lethal disease initiated after the ingestion of Listeria-contaminated food. This species comprises different serovars, from which 4b, 1/2a, and 1/2b cause most of the infections. Among the different proteins involved in pathogenesis, the internalins A (InlA) and B (InlB) are the best characterized, since they play a major role in the enterocyte entry of Listeria cells during early infection. Due to their covalent attachment to the cell wall and location on the bacterial surface, along with their exclusive presence in the pathogenic L. monocytogenes, these proteins are also used as detection targets for this species. Even though huge advancements were achieved in the enrichment steps for subsequent Listeria detection, few studies have focused on the improvement of the antibodies for immunodetection. In the present study, recombinant InlA and InlB produced in Escherichia coli were used as targets to generate antibodies via phage display using the human naïve antibody libraries HAL9 and HAL10. A set of five recombinant antibodies (four against InlA, and one against InlB) were produced in scFv-Fc format and tested in indirect ELISA against a panel of 19 Listeria strains (17 species; including the three main serovars of L. monocytogenes) and 16 non-Listeria species. All five antibodies were able to recognize L. monocytogenes with 100% sensitivity (CI 29.24–100.0) and specificity (CI 88.78–100.0) in all three analyzed antibody concentrations. These findings show that phage display-derived antibodies can improve the biological tools to develop better immunodiagnostics for L. monocytogenes.

Evaluation of the Thermo ScientificTM SureTectTMListeria monocytogenes PCR Assay in a Broad Range of Foods and Selected Environmental Surfaces: Pre-Collaborative and Collaborative Study, First Action 2021.05.

BackgroundThe Thermo Scientific™ SureTect™ Listeria monocytogenes PCR Assay uses Solaris reagents for performing PCR for the rapid and specific detection of Listeria monocytogenes in a broad range of foods and selected environmental surfaces.ObjectiveTo demonstrate reproducibility of the SureTect Listeria monocytogenes PCR Assay in a collaborative study using a challenging matrix, full-fat cottage cheese (25 g). To extend the scope of the method.MethodIn the collaborative study, the candidate method was compared to the United States Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 10 Listeria reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). Eighteen participants from 10 laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Three levels of contamination were evaluated for each matrix. Statistical analysis was conducted according to the probability of detection (POD) statistical model. In addition, to extend the scope, six matrix studies were performed comparing the candidate method to the FDA/BAM reference method. One of these matrixes was also compared to the ISO 11290–1:2017 Microbiology of the Food Chain—Horizontal Method for the Detection and Enumeration of Listeria monocytogenes and of Listeria spp.—Part 1: Detection Method Reference Method.ResultsIn the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated, and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. The two PCR instruments used in the study performed equivalently. All presumptive positives were confirmed via the alternative confirmation procedure. In the pre-collaborative studies, the results showed comparable performances between the candidate method and the reference method for all matrixes tested.ConclusionsBased on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis.HighlightsDue to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 25 g full-fat cottage cheese is known to be a challenging matrix to test. No unusual cross-contamination or false positive/negative data were reported, highlighting the ease of use, reproducibility, and robustness of the method.

Rapid and Specific Detection of Listeria monocytogenes With an Isothermal Amplification and Lateral Flow Strip Combined Method That Eliminates False-Positive Signals From Primer-Dimers.

Listeria monocytogenes is an important foodborne pathogenic bacterium that is explicitly threatening public health and food safety. Rapid, simple, and sensitive detection methods for this pathogen are of urgent need for the increasing on-site testing demands. Application of the isothermal recombinase polymerase amplification (RPA) and the lateral flow strip (LFS) in the detection is promising for fast speed, high sensitivity, and little dependency on equipment and trained personnel. However, the simplicity comes with an intrinsic and non-negligible risk, the false-positive signals from primer–dimers. In this study, an improved RPA–LFS system was established for detection of L. monocytogenes that eliminated false-positive signals from primer–dimers. Primer candidates were carefully selected from the entire L. monocytogenes genome sequence and rigorously screened for specific amplifications in PCR and RPA reactions. For the optimal primer pairs, probes that matched the targeted fragment sequences, although had the smallest chance to form cross-dimers with the primers, were designed and screened. The intelligent use of the probe successfully linked the positive signal to the actual amplification product. This RPA–LFS system was highly specific to L. monocytogenes and was able to detect as low as 1 colony-forming unit of the bacterium per reaction (50 μl) without DNA purification, or 100 fg of the genomic DNA/50 μl. The amplification could be conducted under the temperature between 37 and 42°C, and the whole detection finished within 25 min. Test of artificially contaminated milk gave 100% accuracy of detection without purification of the samples. Various food samples spiked with 10 colony-forming unit of L. monocytogenes per 25 g or 25 ml were successfully detected after an enrichment time period of 6 h. The RPA–LFS system established in this study is a rapid, simple, and specific detection method for L. monocytogenes that has eliminated false-positive results from primer–dimers. In addition, this study has set a good example of eliminating the false-positive risk from primer–dimers in isothermal amplification-based detection methods, which is applicable to the development of detection technologies for other pathogens.

Development and application of Peptide Nucleic Acid Fluorescence in situ Hybridization for the specific detection of Listeria monocytogenes

Listeria monocytogenes is one of the most important foodborne pathogens due to the high hospitalization and mortality rates associated to an outbreak. Several new molecular methods that accelerate the identification of L. monocytogenes have been developed, however conventional culture-based methods still remain the gold standard. In this work we developed a novel Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA-FISH) method for the specific detection of L. monocytogenes. The method was based on an already existing PNA probe, LmPNA1253, coupled with a novel blocker probe in a 1:2 ratio. The method was optimized for the detection of L. monocytogenes in food samples through an evaluation of several rich and selective enrichment broths. The best outcome was achieved using One Broth Listeria in a two-step enrichment of 24 h plus 18 h. For validation in food samples, ground beef, ground pork, milk, lettuce and cooked shrimp were artificially contaminated with two ranges of inoculum: a low level (0.2–2 CFU/25 g or mL) and a high level (2–10 CFU/25 g or mL). The PNA-FISH method performed well in all types of food matrices, presenting an overall accuracy of ≈99% and a detection limit of 0.5 CFU/25 g or mL of food sample.

Screening of Lectins for Specific Detection of Listeria Monocytogenes

Screening of Lectins for Specific Detection of Listeria Monocytogenes

Antibody-aptamer functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes from food

To develop antibody-aptamer functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes from food products. Aptamer, a single-stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer-A8, specific for internalin A, an invasive protein of L. monocytogenes, was used in the fibre-optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti-Listeria antibody, P66, was immobilized on streptavidin-coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647-conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10(3) CFU ml(-1). This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10(2) CFU 25 g(-1) ) ready-to-eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Based on the data presented in this study, the antibody-aptamer functionalized fibre-optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. The study demonstrates feasibility and novel application of aptamer on fibre-optic biosensor platform for the sensitive detection of L. monocytogenes from food products.

A PCR METHOD FOR THE DETECTION OF LISTERIA MONOCYTOGENES BASED ON A NOVEL TARGET SEQUENCE IDENTIFIED BY COMPARATIVE GENOMIC ANALYSIS

ABSTRACTListeria monocytogenes is an opportunistic foodborne pathogen that is a serious health hazard worldwide. In this study, a polymerase chain reaction (PCR) assay was developed for the detection of L. monocytogenes using a novel species‐specific target sequence (a region of lmo0754gene) identified by a comparative genomic approach. An internal amplification control was incorporated into this PCR system. The assay allowed amplification of a 331‐bp fragment only from the genomic DNA of L. monocytogenes strains and not from other Listeria species, as well as some non‐Listeria species. The detection limit of the PCR assay was 55 copies/PCR with L. monocytogenes genomic DNA. Applying this PCR assay to artificially contaminated milk samples, low levels of L. monocytogenes (1–10 cfu/mL of milk) were detected after 6–9 h incubation in selective culture enrichment (UVM1).PRACTICAL APPLICATIONSA novel species‐specific target sequence was identified by a comparative genomic approach that offered a new molecular diagnostic marker for specific detection of Listeria monocytogenes. Using this target, a PCR assay with an internal amplification control was developed and shown to be specific, sensitive and applicable to the detection of L. monocytogenes from artificially contaminated foods. Moreover, this comparative genomic approach could also provide a new tool in mining unique targets for other bacteria with the increasing availability of public data of genome sequences.

Review. Specific detection of Listeria monocytogenes in foods using commercial methods: from chromogenic media to real-time PCR

Listeriosis is one of the most important food-borne diseases. A variety of culture and rapid methods are available for the detection of Listeria spp. in foods. Although the presence of L. innocua may indicate potential contamination with L. monocytogenes, only the latter species is pathogenic for humans. Therefore, the most adequate tests are those which specifically detect L. monocytogenes. Chromogenic media is currently the most common method used for the presumptive identification of L. monocytogenes. Some tests like those based on antigen detection are fast and easily applied, but only a few may specifically detect L. monocytogenes. Real-time polymerase chain reaction is increasingly applied in food diagnostics for the detection of L. monocytogenes due to the availability of different specific commercial test methods. Microarrays and biosensors are some examples of new technologies that might be used routinely for the detection of L. monocytogenes in foods in the future.

Rapid and specific detection of Listeria monocytogenes in smoked salmon with BAX ®-PCR

Members of the genus Listeria are ubiquitous, and are therefore also common to the food and the environment. Among them, only Listeria monocytogenes has a pathogenic potential, and can cause infectious diseases (listeriosis) in humans. Conventional microbiological testing methods are labour-intensive and time consuming (4–5 days), and often require a number of different culture media for final isolation and confirmatory tests. In order to overcome these limitations, numerous rapid methods have been developed in recent years. DNA-based methods such as the polymerase chain reaction (PCR) have increasingly been used for rapid and sensitive detection of L. monocytogenes. Among the various available PCR assays, we used the BAX ® system (with two different detection procedures: gel detection and automated detection) to screen for L. monocytogenes in samples of vacuum packaged cold smoked salmon. A total of 27 samples were used for this study. The method was compared to the German standard microbiological detection method according to DIN 11290-1 and -2. Detection of Listeria and L. monocytogenes from salmon samples was performed using Palcam enrichment medium, followed by plating on both Palcam agar and ALOA agar. The BAX ® assay gave identical results for 26 food samples compared to the standard method, including 15 positives. Only in one case the BAX ® system gave a false-positive result, probably due to the amplification of DNA from nonviable cells of L. monocytogenes. In naturally contaminated food samples, the BAX ® method gave good results after 24–48 h. Application of this rapid method is simple and time saving.

DEVELOPMENT OF A MULTIPLEX PCR ASSAY FOR THE SPECIFIC DETECTION OF SALMONELLA, CAMPYLOBACTER JEJUNI, ESCHERICHIA COLI O157:H7, AND LISTERIA MONOCYTOGENES

Abstract Detection of food‐associated bacterial pathogens has become a major focus of the food industry, regulatory agencies, and researchers. Multiple pathogens need to be rapidly detected with high specificity and as cost efficient as possible. In this research, a multiplex PCR assay for the specific detection of Listeria monocytogenes, Escherichia coli O157:‐H7, Salmonella, and Campylobacter jejuni was developed. The four bacteria were detectable at 104 CFU/PCR reaction. No cross‐reactivity with other bacteria commonly found associated with the four target organisms was found. This assay would simplify detection procedures for the target pathogens, reduce the time and labor necessary to acquire food safety results, and might allow one protocol to be used for bacterial detection on a wide variety of food products.

A PCR protocol using inl gene as a target for specific detection of Listeria monocytogenes

Polymerase chain reaction is a powerful technique for detection of pathogens in foods. It is a rapid procedure with both sensitivity and specificity for quick detection and identification of specific pathogenic bacteria from different sources. Listeria monocytogenes detection methods based on PCR amplification of the iap, prfA and hly gene sequences have been reported. The present study undertakes the development of an alternative PCR method using the inl gene sequences as a target to detect pathogenic L. monocytogenes. The presence of a unique and specific DNA amplification fragment of 760 bp for the intragenic repeats B of the inlA gene in all strains of L. monocytogenes as compared to none in other Listeria and unrelated Gram positive and Gram negative species confirms that this procedure is an alternative PCR protocol for detection of L. monocytogenes.

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.

DNA Extraction and PCR Methods for the Detection of Listeria monocytogenes in Cold-Smoked Salmon

Protocols for the specific detection of Listeria monocytogenes in cold-smoked salmon were developed. PCR was used as the method of detection. Inhibitors of PCR present in the food samples were removed by ether extraction or column purification, or their effect was overcome by the use of Tween 20 as an enhancer. These protocols are many times more rapid than conventional detection methodologies and also have the potential for automation.

Detection of Listeria monocytogenes in surface swabs using a non-radioactive polymerase chain reaction-coupled ligase chain reaction assay

A non-radioactive polymerase chain reaction (PCR)-coupled ligase chain reaction (LCR) assay for the specific detection of Listeria monocytogenes was applied to swab samples from metal surfaces that were artificially inoculated with L. monocytogenes . Using a 24 h enrichment this detection system was able to consistently detect 20 cfu L. monocytogenes from a 25 cm 2 metal surface, also in the presence of 2 × 10 5 cfu of Escherichia coli . Detection of non-viable cells resulting from treatment of the inoculated surface with a chlorinating sanitizer was eliminated by the use of a 24 h enrichment step in Listeria enrichment broth.

Detection of Listeria monocytogenes with a nonisotopic polymerase chain reaction-coupled ligase chain reaction assay.

A polymerase chain reaction (PCR)-coupled ligase chain reaction (LCR) assay for the specific detection of Listeria monocytogenes (M. Wiedmann, J. Czajka, F. Barany, and C. A. Batt, Appl. Environ. Microbiol. 58:3443-3447, 1992) has been modified for detection of the LCR products with a nonisotopic readout. When a chemiluminescent or a colorimetric substrate for the nonisotopic detection of the LCR products was used, the PCR-coupled LCR gave a sensitivity of 10 CFU of L. monocytogenes. The detection method with the chemiluminescent substrate Lumi-Phos 530 permitted detection of the LCR products in less than 3 h, so that the whole assay can be completed within 10 h.

A combined modified reverse dot-blot and nested PCR assay for the specific non-radioactive detection of Listeria monocytogenes

A modified reverse dot-blot assay was developed and used in combination with a nested PCR amplification of hly A for the specific detection of Listeria monocytogenes . The deoxynucleotides and digoxygenin-11-dUTP concentrations in the PCR were optimized for maximal sensitivity and economy. For the dot-blot hybridization, digoxygenin-labelled PCR products were directly spotted on nylon membrane-bound poly-dT tailed capture probes and the signal detection was either colorimetric or chemiluminescent. With crude cell lysates, the total assay could be completed in about 6 h with a detection limit between 2 and 25 colony forming units (cfu) per PCR. The assay was then tested for its applicability to environmental sampling of artificially contaminated aluminum surfaces. For environmental sampling, the assay requires an additional overnight enrichment step and has a sensitivity corresponding to 5 cfu per 25 cm2 of inoculated surface.

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.

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.