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

Abstract

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, membranecompromised 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 10 Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR

Chicken Skin Decontamination of Thermotolerant Campylobacter spp. and Hygiene Indicator Escherichia coli Assessed by Viability Real-Time PCR

“…A v-qPCR for the quantification of viable Thermotolerant Campylobacter spp. was recently validated [42]. To our knowledge, such a publicly available method, which includes a suitable target length and an internal sample process control (ISPC) is still missing for E. coli.…”

“…As usually performed in our Campylobacter-qPCR studies, we quantified using a stable DNA standard with a known chromosomal copy number [42,49,50]. This allowed absolute quantification and yielded approximately 95% amplification efficiency (Figure 2).…”

“…In combination with our previously developed v-qPCR for Thermotolerant Campylobacter spp. [42], we quantified the survival of both C. jejuni and an ESBL-producing E. coli field strain on chicken skin upon the application of different reduction strategies. Theoretically, the sensitivity of the novel qPCR is 100 copies per ml of chicken skin rinse since our standard curve reliably detected 10 copies per PCR reaction and 10% of the DNA was used per sample.…”

“…We prepared DNA quantification standards for the uidA-based qPCR from the E. coli strains DSM 1103 and E. coli ESBL 10,714 as previously described for C. jejuni [42]. In short, strains from liquid BHI cultures were pelleted by centrifugation for 5 min at 16,000× g. The DNA was extracted using the GeneJet Genomic DNA Purification kit according to the manufacturer's manual.…”

“…The novel v-qPCR was applied in parallel with a previously validated v-qPCR for Thermotolerant Campylobacter spp. [42] and CFU determination. They were utilized in order to evaluate the survival of both Gram-negative bacterial fecal pathogens after the application of reduction strategies on chicken skin.…”