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Challenging the “gold standard” of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses

Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Unit 42 Food Hygiene and Food Virology, Germany
Stingl, Kerstin;
Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Unit 42 Food Hygiene and Food Virology, Germany
Heise, Janine;
Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Unit 42 Food Hygiene and Food Virology, Germany
Thieck, Maja;
Affiliation
German Federal Institute for Risk Assessment (BfR), Department 4 Biological Safety, Unit 42 Food Hygiene and Food Virology, Germany
Wulsten, Imke F.;
Affiliation
German Federal Institute for Risk Assessment (BfR), Department of Biological Safety, National Reference Laboratory for Campylobacter, Berlin, Germany
Pacholewicz, Ewa;
Affiliation
Bavarian Health and Food Safety Authority (LGL), Oberschleissheim, Germany
Iwobi, Azuka N.;
Affiliation
Bavarian Health and Food Safety Authority (LGL), Oberschleissheim, Germany
Govindaswamy, Janani;
Affiliation
Bavarian Health and Food Safety Authority (LGL), Oberschleissheim, Germany
Zeller-Péronnet, Véronique;
Affiliation
Bavarian Health and Food Safety Authority (LGL), Oberschleissheim, Germany
Scheuring, Sandra;
Affiliation
National Institute of Veterinary Research (NIVR), Hanoi, Viet Nam
Luu, Huong Quynh;
Affiliation
Institute for Experimental Pathology at Keldur, Reykjavík, Iceland
Fridriksdottir, Vala;
Affiliation
Freie Universitaet Berlin, Institute of Food Safety and Food Hygiene, Berlin, Germany
Gölz, Greta;
Affiliation
BIOTECON Diagnostics GmbH, Potsdam, Germany
Priller, Florian;
Affiliation
University of Ljubljana, Institute of Microbiology and Parasitology, Ljubljana, Slovenia
Gruntar, Igor;
Affiliation
Public Health England, Food, Water and Environmental Laboratory – Porton, Salisbury, United Kingdom
Jorgensen, Frieda;
Affiliation
Wageningen Bioveterinary Research, Lelystad, Netherlands
Koene, Miriam;
Affiliation
The Pennsylvania State University, Department of Food Science, State College, United States
Kovac, Jasna;
GND
1050955064
Affiliation
Max Rubner-Institute (MRI), Department of Safety and Quality of Meat, Kulmbach, Germany
Lick, Sonja;
Affiliation
ANSES, Laboratoire de Ploufragan-Plouzané-Niort, Ploufragan, France
Répérant, Elisabeth;
Affiliation
Impetus GmbH & Co. Bioscience KG, Microbiology, Bremerhaven, Germany
Rohlfing, Annika;
Affiliation
Hirszfeld Institute of Immunology and Experimental Therapy, PAS, Microbiology Department, Wroclaw, Poland
Zawilak-Pawlik, Anna;
Affiliation
State Office for Consumer Protection Saxony-Anhalt, Department of Food Safety, Halle (Saale), Germany
Rossow, Marko;
Affiliation
QuoData Quality & Statistics, Dresden, Germany
Schlierf, Anja;
Affiliation
QuoData Quality & Statistics, Dresden, Germany
Frost, Kirstin;
Affiliation
QuoData Quality & Statistics, Dresden, Germany
Simon, Kirsten;
Affiliation
QuoData Quality & Statistics, Dresden, Germany
Uhlig, Steffen;
Affiliation
Bavarian Health and Food Safety Authority (LGL), Oberschleissheim, Germany
Huber, Ingrid

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the “gold standard” in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log₁₀ Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log₁₀ live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as “gold standard” and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens.

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