TY - JOUR
T1 - Challenging the “gold standard” of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses
AU - Stingl, Kerstin
AU - Heise, Janine
AU - Thieck, Maja
AU - Wulsten, Imke F.
AU - Pacholewicz, Ewa
AU - Iwobi, Azuka N.
AU - Govindaswamy, Janani
AU - Zeller-Péronnet, Véronique
AU - Scheuring, Sandra
AU - Luu, Huong Quynh
AU - Fridriksdottir, Vala
AU - Gölz, Greta
AU - Priller, Florian
AU - Gruntar, Igor
AU - Jorgensen, Frieda
AU - Koene, Miriam
AU - Kovac, Jasna
AU - Lick, Sonja
AU - Répérant, Elisabeth
AU - Rohlfing, Annika
AU - Zawilak-Pawlik, Anna
AU - Rossow, Marko
AU - Schlierf, Anja
AU - Frost, Kirstin
AU - Simon, Kirsten
AU - Uhlig, Steffen
AU - Huber, Ingrid
N1 - Funding 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 .
Funding 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.
Publisher Copyright:
© 2021
PY - 2021/12/2
Y1 - 2021/12/2
N2 - 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.
AB - 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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U2 - 10.1016/j.ijfoodmicro.2021.109417
DO - 10.1016/j.ijfoodmicro.2021.109417
M3 - Article
C2 - 34624596
AN - SCOPUS:85116404744
SN - 0168-1605
VL - 359
JO - International journal of food microbiology
JF - International journal of food microbiology
M1 - 109417
ER -