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Food Science. Listeria monocytogenes. Listeria monocytogenes ; Laboratory manuals. Life Sciences. Life sciences. Listeria monocytogenes pathogenicity. Jordan, Kieran. Related item.

1 Introduction

Other format: ; ISBN: Electronic books. Laboratory Manual. Laboratory manuals. Internet Resources. Summary Listeria monocytogenes is still a major threat to public health. Environmental sampling and testing in processing areas is also required. Food and environmental samples should be tested as soon as possible after collection.

If this is not possible, samples should be stored at 4 o C. The cells survive well in frozen foods, but samples should be kept frozen prior to testing. Composite samples from individual food lots can be prepared by blending samples in enrichment broth media. Typically, 25g food samples are cultured in detection testing and duplicate samples should be retained for enumeration if a detection test gives a positive result. There are current ISO horizontal methods for the detection and enumeration of L. ISO is a method for the detection of L.

Both methods were amended in to include modified media. The first stage in traditional detection methods for food and environmental samples is usually a pre-enrichment culture in a liquid medium such as Buffered Listeria Enrichment Broth BLEB , or Half-Fraser broth, incubated at 30 o C for 24 hours. Pre-enrichment media may contain selective agents to inhibit the growth of competing micro-organisms, but usually at lower concentrations than those used in selective enrichments. The pre-enrichment culture is than subcultured into selective enrichment media, such as Fraser broth and Oxoid Novel Enrichment ONE broth - Listeria , and incubated for a further 24 hours at o C.

The selective enrichment culture is inoculated on to one or more selective agar media and incubated at 37 o C for hours. A variety of alternatives are available, including Modified Oxford agar, Palcam agar and LPM agar, all of which utilise aesculin hydrolysis by Listeria spp. Several selective chromogenic agar media specifically designed for the differentiation of L.

Where detection methods show a positive result for Listeria spp. Typically, this is done by preparing sample dilutions in buffered peptone water, or enrichment broth without supplements, plating each dilution onto ALOA, or another selective agar medium, and incubating at 37 o C for hours.

Typical colonies can then be counted and confirmed. Listeria spp. Some methods can be automated to screen large numbers of samples. Almost all rapid test protocols include a selective enrichment culture, sometimes shortened to 24 hours, and then apply rapid detection techniques to replace culture on selective agars and further confirmatory tests. Most can claim to produce a result in around half the time taken by traditional methods and certain PCR-based assays are capable of detecting Listeria within 30 hours. Preliminary identification based on colony appearance on chromogenic and other selective agar media is traditionally confirmed using classical biochemical and morphological tests.

Another useful confirmatory test for Listeria spp is the Christie-Atkins-Munch-Peterson CAMP test, which can help confirm species by testing for haemolysis enhancement on sheep blood agar in the presence of other haemolytic bacteria. Listeria isolates can be further characterised by serological typing. Typing can be important in investigating foodborne disease outbreaks and in tracking individual strains of L. Immunological identification and confirmation tests based on latex agglutination, enzyme immunoassay EIA and enzyme-linked immunosorbent assay ELISA have been developed for Listeria spp.

Like to get the latest updates on food pathogen detection products sent to your email? Subscribe to the rapidmicrobiology eNewsletter. This site works best with JavaScript enabled. What is Listeria monocytogenes? Bacteria of the genus Listeria are Gram-positive, facultatively anaerobic, non-spore forming rods and are common in the natural environment. The genus currently contains six species, L. Four species have been implicated in cases of human illness, but L. The species is made up of at least 13 different serotypes, all of which can cause listeriosis, but the most commonly found in human infection is serotype 4b.

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How do I Screen Foods for Listeria? Further evaluation with five other Listeria spp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity.


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This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. Listeria monocytogenes is one of six species within the Listeria genus, with the other six identified as L. They are Gram positive, facultative anaerobic, non-spore-forming, rod-shaped bacteria 0. Although L. Listeria spp. Although the incidence rate of L.

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There are thirteen different L. The current commercially available tests for Listeria spp. To enhance the detection and identification of L. A number of rapid methods have been developed, for example immunological techniques such as biosensor [ 10 , 11 ], enzyme-linked immunosorbent assay ELISA [ 12 ] and antibody array [ 13 , 14 ] and, genotypic techniques such as real-time polymerase chain reaction RT-PCR [ 15 — 17 ], deoxyribonucleic acid DNA microarray [ 18 ] and loop-mediated isothermal amplification LAMP methods [ 19 , 20 ]. Although genotypic techniques that rely on detecting unique DNA sequences between species are available, such techniques can be time-consuming requiring additional sample extraction steps.

Culture combined with immunological techniques are used more routinely, however, generating antibodies with the desired specificity and sensitivity for L.

Listeria monocytogenes

Successful rapid diagnostic techniques require a specific and high affinitybinder, usually an antibody. Development of animal derived antibodies is not an easy task and they are expensive to produce. Phage display biopanning is a technique extending from the invention of phage display technology in [ 22 ] and offers an alternative means of generating specific affinity ligands.

This technique has been used previously in an attempt to generate alternative binders to L. However, Carnazza et al.

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This study aimed to use phage display biopanning to produce L. The biopanning regime employed subtraction biopanning against L. The ultimate goal was to determine the potential utility of the phage display-derived L. Target antigen for positive surface biopanning was L. Each culture was standardized to 2x10 9 cfu ml -1 in phosphate buffered saline pH 7. Once irradiated the cultures underwent a fold concentration with final resuspension in 0. The Salmonella spp.

Four surface biopannings Figure 1 were performed using a phage display peptide library [Ph. The first three rounds were performed against L. The fourth included an initial subtraction biopanning step performed against L. Two solution biopannings Figure 1 were performed following the completion of the third surface biopanning round and the protocol based on a combination of the surface biopanning protocol in the NEB kit instructions and the solution biopanning protocol employed by Paoli et al.

Each round included one solution biopanning against viable L. The cultures were prepared and standardized to 2x10 9 cfu ml -1 in the same manner as described above and the bacterial cells washed twice in tris buffered saline pH 7. Four sterile 1. Eppendorf 2 was centrifuged and the pellet of phages bound to L. Eppendorf 3 was centrifuged, the supernatant containing phages bound to L. This unamplified phage from solution biopanning round 4B was tittered and amplified.

This solution biopanning protocol was repeated one additional time round 5. The SAROTUP tool [ 28 ] was used to ensure deciphered peptide sequences were not previously recognised target-unrelated peptide sequences [ 29 ]. Unique peptide sequences were screened for their ability to bind to L. Stock cultures of L. On the basis of the phage binding ELISA results the mer peptides expressed by eight phage clones showing greatest capability to bind L. Table 3.

Information on peptides chemically synthesised and evaluated for specific binding to L. Protein concentration was measured at nm for post-coupling supernatants from the preparation of peptide-coated beads to verify that coupling had occurred.

Listeria monocytogenes

Serial dilutions fold of L. The MS protocol used peptide-coated beads on duplicate aliquots of two cell concentrations of L. The concentrations of 3x10 2 and 3x10 3 cfu ml -1 were chosen because a countable number of colonies would result if capture occurred. The plate was washed as described above before goat anti-mouse horseradish peroxidase final concentration of 0. For sensitivity studies the same ELISA protocol was used with a total of 11 bacterial concentrations half-log increments from 1x10 3 -1x10 8 cfu ml The intensities were used to fit on a dose—response curve with equation [ 14 , 30 ]:.

Y is the intensity when detecting bacteria at concentration X, while A, B, and C are constants obtained from the curve fitting.


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The limit of detection LOD was calculated using the intensity values greater than twice the background [ 31 , 32 ]. Seventeen phage clones were selected from throughout the biopanning series for more extensive evaluation by phage binding ELISA Table 4. Eight of the 17 had their equivalent synthetic peptides previously evaluated by MS and sandwich ELISA Table 3 and the other nine were from solution panning round 5. For the specificity study the phage clones were used at 1x10 11 pfu ml -1 in PBS However, for these experiments viable bacteria L. Each phage clone evaluation was performed in duplicate.

Based on the fold difference results the four best L. Table 4. Information on phage clones from biopanning rounds 3, 4A and 5 chosen for further evaluation in terms of specificity for L. For the sensitivity experiment the same four phage clones were evaluated in the same manner as for the specificity experiment with serial concentrations 10 3 8 cfu ml -1 of viable L.

The experiment was performed in duplicate and LOD values for each phage clone were calculated as described above. The results of biopanning are summarised in Table 2. A after surface biopanning round 3 B after surface biopanning round 4A and C after solution biopanning round 5. Each experiment performed once only due to limited amount of phage clone stock available for testing. Normalized data equates to relative OD signal after subtraction of background OD signal.