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 log io 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 logio 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.
Bibliographical noteFunding Information: This study was funded by the Federal Ministry of Education and Research (BMBF), project CAMPY-TRACE, 031B0054A/B , PTJ-BIO. We thank our members of the NRL for technical support. HQL received funding from the Ministry of Science and Technology (MoST) of Vietnam project No. NĐT.12.GER/16 .
Open Access: This is an open access article under the CC BY-NC-ND license.
Publisher Copyright: © 2021 The Authors. Published by Elsevier B.V.
Citation: Kerstin Stingl, Janine Heise, Maja Thieck, Imke F. Wulsten, Ewa Pacholewicz, Azuka N. Iwobi, Janani Govindaswamy, Véronique Zeller-Péronnet, Sandra Scheuring, Huong Quynh Luu, Vala Fridriksdottir, Greta Gölz, Florian Priller, Igor Gruntar, Frieda Jorgensen, Miriam Koene, Jasna Kovac, Sonja Lick, Elisabeth Répérant, Annika Rohlfing, Anna Zawilak-Pawlik, Marko Rossow, Anja Schlierf, Kirstin Frost, Kirsten Simon, Steffen Uhlig, Ingrid Huber, Challenging the “gold standard” of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses, International Journal of Food Microbiology, Volume 359, 2021, 109417, ISSN 0168-1605,
- Food safety
- Interlaboratory validation
- Propidium monoazide