European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.
The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy. Electronic address: monica.gianfranceschi@iss.it.
Monica Virginia
MV
Gianfranceschi
Instituto Tecnológico Agrario de Castilla y León (ITACyL), Valladolid, Spain; Microbiology Section, Faculty of Sciences, University of Burgos, Burgos, Spain.
David
D
Rodriguez-Lazaro
Instituto Tecnológico Agrario de Castilla y León (ITACyL), Valladolid, Spain.
Marta
M
Hernandez
Instituto Tecnológico Agrario de Castilla y León (ITACyL), Valladolid, Spain.
Patricia
P
González-García
Istituto Zooprofilattico Sperimentale delle Venezie, SCS 8 - Valorizzazione delle Produzioni Alimentari, Padua, Italy.
Damiano
D
Comin
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.
Antonietta
A
Gattuso
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.
Elisabetta
E
Delibato
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.
Michele
M
Sonnessa
Alma Mater Studiorum, Università di Bologna, Dip. Scienze e Tecnologie Agro-Alimentari Ozzano dell'Emilia (BO), Italy.
Frederique
F
Pasquali
National Reference Laboratory Listeria monocytogenes, Istituto Zooprofilattico Spermentale degli Abruzzi e Molise, Teramo, Italy.
Vincenza
V
Prencipe
National Food Chain Safety Office, Food and Feed Safety Directorate, Food Microbiological National Reference Laboratory, Mester, Budapest, Hungary.
Zuzsanna
Z
Sreter-Lancz
CNTA, Centro Nacional de Tecnología y Seguridad Alimentaria, San Adrian, Navarra, Spain.
María-José
MJ
Saiz-Abajo
CNTA, Centro Nacional de Tecnología y Seguridad Alimentaria, San Adrian, Navarra, Spain.
Javier
J
Pérez-De-Juan
CNTA, Centro Nacional de Tecnología y Seguridad Alimentaria, San Adrian, Navarra, Spain.
Javier
J
Butrón
University of Zagreb, Faculty of Veterinary Medicine, Dpt. of Hygiene, Technology and Food Safety, Zagreb, Croatia.
Lidija
L
Kozačinski
University of Zagreb, Faculty of Veterinary Medicine, Dpt. of Hygiene, Technology and Food Safety, Zagreb, Croatia.
Danijela Horvatek
DH
Tomic
University of Zagreb, Faculty of Veterinary Medicine, Dpt. of Hygiene, Technology and Food Safety, Zagreb, Croatia.
Nevijo
N
Zdolec
Section for Bacteriology - Food and GMO, Norwegian Veterinary Institute, Oslo, Norway.
Gro S
GS
Johannessen
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Džiuginta
D
Jakočiūnė
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.
John Elmerdahl
JE
Olsen
Direzione Operativa Controllo degli Alimenti, Istituto Zooprofilattico Sperimentale del Lazio e Toscana, Rome, Italy.
Paola
P
De Santis
Direzione Operativa Controllo degli Alimenti, Istituto Zooprofilattico Sperimentale del Lazio e Toscana, Rome, Italy.
Sarah
S
Lovari
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy.
Barbara
B
Bertasi
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy.
Enrico
E
Pavoni
Istituto Zooprofilattico Sperimentale delle Venezie, SCS 8 - Valorizzazione delle Produzioni Alimentari, Padua, Italy.
Antonella
A
Paiusco
Alma Mater Studiorum, Università di Bologna, Dip. Scienze e Tecnologie Agro-Alimentari Ozzano dell'Emilia (BO), Italy.
Alessandra
A
De Cesare
Alma Mater Studiorum, Università di Bologna, Dip. Scienze e Tecnologie Agro-Alimentari Ozzano dell'Emilia (BO), Italy.
Gerardo
G
Manfreda
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.
Dario
D
De Medici
D002611:Cheese / Q000382:microbiology*; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000235:genetics; D005060:Europe; D005516:Food Microbiology / Q000379:methods*; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial
Artificial contamination; ISO 11290-1:1996+Amd.1:2004; Listeria monocytogenes; Real time PCR; Soft cheese; Validation