## 4 Discussion
### Jejuni and C. coli
[46] showed the critical impact of droplet volume variability in ddPCR on the accuracy of the absolute quantification. The applicability of our v-ddPCR to quantify _Campylobacter_ was investigated on natural contaminated routine samples from LGL, comprising chicken neck skin from slaughterhouses as well as breast meat and naturally contaminated retail samples, purchased in Bavarian supermarkets. The samples were analyzed in parallel with the reference method ISO 10272-2:2017 [7] and v-qPCR [18]. [PERSON] et al. [43] pointed out the characteristic and the unique potential of ddPCR technology to provide an absolute quantitative value of a specific target, but meanwhile there was a difficulty in verifying against other molecular methods. The used v-qPCR method relies on a calibration curve, delivering a quantitative value and thus confirming the accuracy of our ddPCR results when analyzing naturally contaminated samples. Solely, chicken neck skin samples from slaughterhouses (around 50% of analyzed samples) were quantified above PHC (3.0 log\({}_{10}\) CFU/g chicken meat) on the reference plate count method; all breast meat and retail samples were detected under PHC on mCCD plates. The EU Regulation No 2017/1495 [6] already highlighted the existence of different significant contamination levels between neck skin and breast skin samples. Chicken neck skin samples from slaughterhouses have been shown to be much more contaminated than breast meat samples (skin-free) or retail samples from the supermarkets that are usually exposed to a long and stressful cooling of the refrigerated counter [19]. Except for NS-20, the plate results of chicken neck skin were confirmed with both alternative PCR methods, which revealed a good correlation of both PCR methods with the official plate count method for the slaughterhouse samples. For two neck skin samples, NS-19 and NS-35, _Campylobacter_ could be quantified above the PHC only in a v-qPCR, and it remained undetected by the plate count method, as well as in the v-ddPCR. It cannot be excluded that another thermophilic _Campylobacter_ spp. (e.g., _C. lari_) is involved, which could not be detected in a v-ddPCR method, which used other detection systems as v-qPCR and only detects _C. jejuni_ and _C. coli_. This may also be related to the sensitivity of the different detection systems. While v-ddPCR is based on specific single-copy genes for _C. jejuni_ and _C. coli_, three target copies of the 16S rRNA gene per chromosomal copy in _Campylobacter_ spp. may be detected in v-qPCR [18]. Out of twenty-five retail samples, two samples--RS-03 and RS-15--were quantified above the PHC limit for both molecular methods; one additional sample, RS-04, was quantified above PHC only in v-ddPCR. The ISO standard method failed to quantify these three samples above PHC. _Campylobacter_ may have lost its cultivability due to oxygen or cold stress during the 4 \({}^{\circ}\)C storage in supermarkets. These potentially infectious _Campylobacter_ cells in the VBNC stage could not be microbiologically detected as CFU, so the level of contamination of poultry meat is clearly underestimated [47], and it represents a significant threat to human health. Hence, the current duplex v-ddPCR assay can be helpful for risk assessment of _Campylobacter_ in broiler meat, especially for the previously cooled retail samples. Finally, although we observed an acceptable correlation for quantifying _Campylobacter_ between the three applied methods, none of the tested methods was successful in detecting all the _Campylobacter_ positive samples beyond the PHC. Independent of the PHC, v-ddPCR could detect more _Campylobacter_ positive samples (49%) than the other methods (v-qPCR 33%, plate count 40%) among the total number of samples. Worldwide, scientists have reported a higher sensitivity of ddPCR compared to qPCR [29; 45]. The ddPCR partitions every sample into thousands of droplets, and it performs independent end-point PCR amplification on each droplet.