## 4 Discussion  
### Jejuni and C. coli  
are the predominant _Campylobacter_ species in poultry causing the most foodborne zoonosis and leading to a real impact on public health care. A review based on novel microbial risk assessment studies [5] like QMRA (Quantitative Microbial Risk Assessments) and based on CFU data shows the relationship between the prevalence of _Campylobacter_ spp. in the poultry meat chain (broiler flocks, slaughterhouses, and consumers of poultry meat), and public health infection risk. The official ISO 10272-2:2017 method [7] used for the enumeration of _Campylobacter_ spp. in poultry meat is unable to cultivate all infectious bacteria (IPTU), including CFU and VBNC, and it requires a long (2 days) and laborious (microaerobic atmosphere) incubation. Moreover, we observed the overgrowth of accompanying bacterial flora during the analysis of slaughterhouse samples, even on _Campylobacter_-selective mCCD agar plates, as well as samples with high levels of _Campylobacter_ contamination make the microbiological enumeration of _Campylobacter_ spp. unreliable for its use in routine analysis. A viability PMA-qPCR (v-qPCR) has been recently published [18], which overcomes these limitations, and it can be successfully applied for specific and sensitive quantification of _Campylobacter_ spp. in poultry. Digital PCR, the third generation of PCR technology, offers further advantages, such as an absolute quantification without an external calibration curve. Therefore, the final result is not influenced by the standard setting. In addition, due to the enrichment effect of the target of interest in the interfering background, ddPCR is highly tolerant to PCR inhibitors [28; 45; 46]. Applying a method comparison study according to ISO 16140-2:2016 [30], we internally validated a PMA-based duplex ddPCR method (v-ddPCR) against the official ISO 10272-2:2017 method [7] in order to quantify viable C. _jejuni_ and _C. coli_ in poultry meat of slaughterhouse and retail samples. The method was supposed to be practicable and cost-effective for routine analysis. On average, no consistent bias was observed between both  
Figure 6: Analysis of different raw chicken (RS) meat samples from retail supermarket on culture, v-ddPCR and v-qPCR method. The Process Hygiene Criterion (PHC: \(\log_{10}\) 3.0) is marked with a black line. methods during the whole trueness study. Likewise, no detectable systematic bias was observed during the accuracy study for _C. coli_, in contrast, a slight positive bias (v-ddPCR against reference method) was noticed for _C. jejuni_. Finally, all criteria from ISO 16140-2:2016(E) [30] were fulfilled for both accuracy and relative trueness validation. The Process Hygiene Criterion of maximum 1000 CFU _Campylobacter_/g chicken skin (1:10 dilution corresponds to 6.7 cp/reaction (0.8 log\({}_{10}\) cp/reaction) and 1:2 dilution corresponds to 33 cp/reaction (1.5 log\({}_{10}\) cp/reaction)) can be reliably detected with ddPCR, as the limit of detection is 4.2 cp/reaction (0.6 log\({}_{10}\) cp/reaction) for _C. jejuni_ and 5.7 cp/reaction (0.8 log\({}_{10}\) cp/reaction) for _C. coli_. The evaluation of the limit of quantification (LOQ) was based on a precision experiment, and it reached 42 cp/reaction (1.6 log\({}_{10}\) (cp/reaction) for _C. jejuni_ and 16 cp/reaction (1.2 log\({}_{10}\) (cp/reaction) for _C. coli_ at RSDr 25%. We recommend, as reported by [PERSON] [22], to increase the number of technical replicates analyzed in ddPCR so that the LOQ can be significantly decreased and the PHC can be reliably quantified (see Figure 1a,b). Likewise, the range of the technical uncertainty reaches a better performance if two technical replicates are included in the analyses (see Table 11). Furthermore, based on the uncertainty data we believe that a higher repeatability and reproducibility (in particular: lower factorial effects) can be achieved by better standardization of the method. Additionally, [PERSON] et al.