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

Bohnert, Markus; Dilasser, Françoise; Dalet, C.; Mengaud, J; Cossart, Pascale

doi:10.1016/0923-2508(92)90019-k

Cited by 53 publications

(45 citation statements)

References 24 publications

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“…In the present study, hlyA and inlA in twent L. monocytogenes were positive, which is more than other report (91.7%) (20). The positive CAMP and haemolysis assay and the presence of hlyA and inlA genes by PCR in all positive isolates suggest that these virulent strains may have the potential to invade host cells and cause listeriosis (21). The results obtained have confirmed that the environment is a source of potentially pathogenic L. monocytogenes strains that carry a basic set of virulence genes and have the potential for invading the host epithelial cells.…”

Section: Discussionsupporting

confidence: 70%

Isolation of Listeria monocytogenes of Karun River (Enviromental Sources Rural and Urban) by Culture and PCR Assay

Nassirabady¹,

Meghdadi²,

Alami³

2015

Int J Enteric Pathog

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Background: Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. Listeriolysin O (LLO) and internalin A are two major pathogenesis factors in this bacterium. Objectives: The purpose of this research was to isolate of Listeria from different parts of the Karun river in Iran. The bacteria were identified based on cultural characteristics, biochemical tests and by PCR assay. Materials and Methods: A total of 150 water samples from Karun river (rural and urban environment) were collected. The bacteria were identified based on cultural characteristics, biochemical tests and by PCR assay. Results: From total 150 samples, twenty L. monocytogenes were isolated and identified and amplification of two pathogenicity genes: hlyA and inlA in twenty L. monocytogenes were positive. Conclusions: Detection of Listeria monocytogenes with hlyA and inlA genes suggest that these strains may have the potential to invade host cells, and consumption of water contaminated with L. monocytogenes, can cause human disease.

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Section: Discussionsupporting

confidence: 70%

Isolation of Listeria monocytogenes of Karun River (Enviromental Sources Rural and Urban) by Culture and PCR Assay

Nassirabady¹,

Meghdadi²,

Alami³

2015

Int J Enteric Pathog

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“…In the present work we compared two molecular detection methods, one immunoenzymatic and the other PCR based, specific for L. monocytogenes with the standard culture method ISO 11290-1. Studies on the application of PCR detection in naturally contaminated food are very scarce, but nevertheless the reliability and stability of the PCR approach compared with culture methods has been underlined (Bohnert et al, 1992;Niederhauser et al, 1992Niederhauser et al, , 1993. In addition, as we have demonstrated before (Aznar and Alarcón, 2003), there are multiple factors affecting the sensitivity of PCR detection of L. monocytogenes which complicates the comparison with the results obtained in other works.…”

Section: Discussionmentioning

confidence: 48%

PCR Detection of Listeria monocytogenes in different Food Products compared with the mini-VIDAS LMO System and the Standard Procedure ISO 11290-1

Aznar

Solís²

2006

J. Verbr. Lebensm.

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The presence of Listeria monocytogenes in 225 natural samples, including different food types, was investigated by three methods: (i) culture-based standard procedure ISO 11290-1, (ii) mini-VIDAS (Vitek Immuno Diagnostic Assay System) LMO, an enzyme linked fluorescent assay (ELFA) commercially available, and (iii) PCR using a previously established procedure. Identification of isolates recovered from the standard method and mini-VIDAS, on Oxford and PALCAM selective plates, was carried out using the API-Lis system and also by PCR, using L. monocytogenes specific primers. In all, 65 samples (64 meat products and one smoked salmon) were positive with any of the three procedures assayed. Taking into account the results based on PCR identification, the standard culture-based method found 22 and the mini-VIDAS 23, while PCR detected 60 positive samples in a total of 225 food samples. For comparative purposes, a set of "known positive samples" was established as reference that included 33 natural samples from which L. monocytogenes isolates (identified by specific PCR) had been recovered. The mini-VIDAS and ISO 11290-1 methods were equally sensitive. Compared to them PCR was clearly the more accurate and efficient procedure for detection of L. monocytogenes in food. Moreover, it showed no false negative results. The PCR approach can be completed in 48 working hours, and because of its specificity might eventually be cheaper than the other two procedures. Consequently we recommend PCR for routine detection of L. monocytogenes in food. Zusammenfassung(Redaktion): 225 Lebensmittelproben wurden auf das Vorhandensein von Listeria monocytogenes mit Hilfe von drei verschiedenen Methoden untersucht: (i) durch die auf Kultivierung basierende Standard-Methode nach ISO 11290-1, (ii) durch mini-VIDAS (Vitek Immuno Diagnostic Assay System) LMO, einem Enzym-basierten Verfahren mittels Fluoreszenz-Nachweis (ELFA; im Handel erhältlich) und (iii) durch ein PCR-Verfahren, das jüngst von den Autoren etabliert worden ist. Die Identifikation von Isolaten durch Einsatz der Standard-oder der mini-VI-DAS-Methode wurde nachvollzogen durch Verwendung des API-Lis-Systems und auch durch die PCR unter Verwendung von L. monocytogenes-spezifischen Primern. Insgesamt wurden 65 Lebensmittelproben durch jede der drei Methoden als positiv identifiziert. Durch die Standard-Methode wurden 22, durch die mini-VIDAS 23 und durch die PCR 60 positive Proben unter den 225 Lebensmittelproben identifiziert. Zu Vergleichszwecken wurden Referenzproben (belastet mit L. monocytogenes) durch PCR positiv identifiziert.Das mini-VIDAS-und das Verfahren nach ISO 11290-1 waren von gleicher Sensitivität; aber im Vergleich zu ihnen war die PCR deutlich exakter und effizienter, um L. monocytogenes in Lebensmitteln nachzuweisen. Das Ergebnis des Nachweises mittels PCR kann nach 48 Arbeitsstunden vorliegen. Daher empfehlen die Autoren ihre PCR als Routine-Verfahren für den Nachweis von L. monocytogenes in Lebensmitteln.

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“…After centrifugation (10 min at 10 000 g), 5 µL of the supernatant was subjected to PCR. PCR amplification of a fragment internal to hly of 388 bp, the Listeria monocytogenes gene coding for listeriolysin O, was performed using primers G0 (5' GAA-TGT-AAA-CTT-CGG-CGC-AAT-CAG-3') and D0 (5' GCC-GTC-GAT-GAT-TTG-AAC-TTC-ATC-3'), shown by Bohnert et al [7] to be specific for Listeria monocytogenes. The reaction mixture contained 10× PCR buffer, 2.5 mM of each dNTP, 25 µM of each primer, 5 µL of the DNA preparation and 0.1 U of HotGoldStar DNA polymerase (Eurogentec) in a total volume of 50 µL.…”

Section: Mouse Virulence Assaysmentioning

confidence: 99%

Avirulence of Viable But Non-Culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

et al. 2005

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-The virulence of Viable But Non-Culturable (VBNC) cells of 4 strains of Listeria monocytogenes was investigated in both a human adenocarcinoma cell line (HT-29) and a mouse model. LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in microcosm water at 20 °C and Scott A strain at 4 °C. No culturable bacteria were detected in the VBNC state, although 10 4 active cells/mL were found by the Direct Viable Count (DVC) and CTC-DAPI double staining methods. A comparison of virulence in both human adenocarcinoma cell line HT-29 and the mouse model showed that culturable controls were more virulent than VBNC cells, which appeared to be avirulent regardless of the virulence methods applied. Pathogenicity was tested in each model and was lost concomitantly with culturability, whereas some cells were still metabolically active (determined by CTC and DVC). Moreover, amplification of a 388 bp fragment with Immunocapture-PCR revealed the presence of Listeria monocytogenes DNA in all mixed spleen samples after intravenous injection of VBNC cells. These results demonstrate that VBNC cells were present in the mouse spleens. The results of the study suggest that Listeria monocytogenes strains might remain in the aquatic environment for prolonged periods in the VBNC state but these cells were not pathogenic in the conditions tested. These findings demonstrate the value of VBNC studies and show the need to investigate the role of VBNC cells in environmental transmission of Listeria monocytogenes. Further studies are needed in order to investigate the virulence of VBNC cells of Listeria monocytogenes after recovery of a culturable state. VBNC / virulence / Listeria monocytogenes / plaque forming assay

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

Cited by 53 publications

References 24 publications

Isolation of Listeria monocytogenes of Karun River (Enviromental Sources Rural and Urban) by Culture and PCR Assay

Isolation of Listeria monocytogenes of Karun River (Enviromental Sources Rural and Urban) by Culture and PCR Assay

PCR Detection of Listeria monocytogenes in different Food Products compared with the mini-VIDAS LMO System and the Standard Procedure ISO 11290-1

Avirulence of Viable But Non-Culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

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