# Inter-Laboratory Validation Trial on a multiplex real-time PCR method allowing the identification of 30 major Clonal Complexes of Listeria monocytogenes circulating in food in Europe

Fabrizia Guidi, Karine Capitaine, Benjamin Felix

► To cite this version:

Fabrizia Guidi, Karine Capitaine, Benjamin Felix. Inter-Laboratory Validation Trial on a multiplex real-time PCR method allowing the identification of 30 major Clonal Complexes of Listeria monocytogenes circulating in food in Europe. Food micro 2024, Jul 2024, Burgos (Spain), France. hal-05524026

HAL Id: hal-05524026

https://hal.science/hal-05524026v1

Submitted on 23 Feb 2026

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
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anses

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EURL Lm

European Union Reference
Laboratory for

Listeria monocytogenes
http://eurl-listeria.anses.fr

IZS
TERAMO

ISTITUTO
ZOOPROFILATTICO
SPERIMENTALE
DELL'ABRUZZO
E DEL MOLISE
"G. CAPORALE"

Organized by

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International Committee
on Food Microbiology
and Hygiene

Under the auspices:

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UNIVERSIDAD
DE BURGOS

F
OD
mcr
2024

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TECHNOLOGICAL EVOLUTION
AND REVOLUTION IN FOOD
MICROBIOLOGY
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TECHNOLOGICAL EVOLUTION
AND REVOLUTION IN FOOD
MICROBIOLOGY

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anses

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EURL Lm

European Union Reference
Laboratory for

Listeria monocytogenes
http://eurl-listeria.anses.fr

IZS
TERAMO
/

ISTITUTO
ZOOPROFILATTICO
SPERIMENTALE

DELL'ABRUZZO
E DEL MOLISE
"G. CAPORALE"

Organized by:

<figure><img src="image_13.png" /></figure>

International Committee
on Food Microbiology
and Hygiene

Under the auspices:

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UNIVERSIDAD
DE BURGOS

Inter-Laboratory Validation Trial on a multiplex real-time
PCR method allowing the identification of 30 major
Clonal Complexes of *Listeria monocytogenes*
circulating in food in Europe

Fabrizia Guidi / IZSAM

f.guidi@izs.it
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Materials and methods

Zenodo

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Standard
Operating
Procedure

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Identification by High-Throughput Real-Time PCR of 30 Major
Circulating *Listeria monocytogenes* Clonal Complexes in Europe

© Benjamin Félix,ª Karine Capitaine,ª Sandrine Te,ª Arnaud Felten,ᵇ Guillaume Gillot,ᶜ Carole Feurer,ᵈ Tijs van den Bosch,ᵉ

© Marina Torresi,¹ Zsuzsanna Sréterné Lancz,ᵇ Sabine Delannoy,ᵇ Thomas Brauge,¹ Graziella Midelet,¹ Jean-Charles Leblanc,ª
Sophie Rousselª

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Published

- 98 blinded pure strains of Listeria

**CC PCR assessment:**

90 strains had to be correctly identified and 8 strains (4 Lm and 4 L. spp) negative

**Molecular serotyping PCR assessment:**

20 strains were taken from the 98 strains panel

- Provided by coordinating laboratory: primers and hydrolysis probes

- At convenience of the participants: DNA extraction method, master mix and thermocycler
Inter-laboratory validation trial - Results

<table><caption>Table 1: Results of the 15 laboratories in 11 countries</caption><thead><tr><th></th><th>For CC PCR</th><th>For molecular serotyping</th></tr></thead><tbody><tr><td>Overall concordance</td><td>99.2%</td><td>100%</td></tr><tr><td>Individual concordance</td><td>93.9% (92/98) to 100%</td><td>100%</td></tr><tr><td>Major issue</td><td>Master mix compatibility</td><td>-</td></tr></tbody></table>

<figure><img src="image_20.png" /><figcaption>Figure 1: map of the 15 participating laboratories</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>Figure 2: CC PCR cycle threshold values for each laboratory. Red line shows the PCR positivity limit. Coloured items depend on lab designation on the map.</figcaption></figure>
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# Discussion - Perspectives

- Comparable to former PCR ILVT (Doumith et. al. 2004 JCM)

- Master mixes with issues will be exclude from the new version of the method, master mixes without issue will be recommended

- Crucial advantages compared to MLST: rapid, reproducible, cost effective, pivotal for outbreak strain screening

- Reliability of the 35 real-time PCR

- Helpful for EU-wide investigation (Costa et. Al. 2022, EFSA)

- Publication of the results - Microbiologie Spectrum - in progress

- Application on complex matrices - in progress

# Co-authorship

**ILVT study group:**

Marina Torresi, National reference Laboratory for Listeria monocytogenes, Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. Caporale - Italy

Hana Plodková, State Veterinary Institute - Czech Republic

Stefanie Lueth, German Federal Institute for Risk Assessment - Germany

Tone Mathisen Fagereng, Taran Skjerdal, Norwegian Veterinary Institute - Norway

Monica Ricão, Paula Ágren, Swedish Food Agency - Sweden

Zsuzsanna Sréterné Lancz, Microbiological National Reference Laboratory, National Food Chain

Safety Office - Hungary

Angela van Hoek, Wilma Jacobs-Reitsma, National Institute for Public Health and the Environment (RIVM) - The Netherlands

Hugo Guedes, Gonçalo Nieto Almeida, INIAV National Institute for Agricultural and Veterinary Research (INIAV) - Portugal

Carole Feurer, Baptiste Houry, IFIP-The French Pig and Pork Institute, Department of Fresh and Processed Meat - France

Valérie Michel, Pauline Sabrou, ACTALIA Département Microbiologie Laitière - Unité Expertise analytique Laitière - France

Thomas Brauge, Cécile Dumaire, ANSES, Laboratory for Food Safety, Bacteriology and Parasitology of Fishery and Aquaculture Products Unit - France

Erwan Bourdonnais, ACTALIA, Food Safety Unit - France

Laurentiu Mihai Ciupescu, Institute of Hygiene and Veterinary Public Health - Romania

Authors' only andごacceptance required those of the European Union or European Health and Digital Executive Agency (HaDEA). Neither the European Union nor HaDEA can be held responsible for those of the Gillot, Nassim Mouhali, ADRIA Food Technical Institute - France