## 4 Confirmation and Identification of _Campylobacter_ spp.
### Molecular Methods for Differentiating Campylobacter Species  
The inherent disadvantage that biochemical differentiation of _C. jejuni_ and _C. coli_ has because of the low specificity (_ca._ 20%) hippurate hydrolysis exhibits to some hippurate-negative strains of _C. jejuni_[122; 123], together with the laborious and time-consuming character of biochemical identification tests, stimulated the development of molecular diagnostic methods and techniques as alternatives to the classical culture-dependent approach to differentiation of _Campylobacter_ species.  
PCR-Based Methods and Techniques  
The technique of mPCR has been widely studied by several researchers in an effort to find a fast and at the same time reliable means of identifying isolated _Campylobacter_ species [142; 143; 144]. The combination of food sample enrichment with multiplex real-time PCR (mrt-PCR) results in more rapid detection and identification of _Campylobacter_ spp. isolated from food [145], compared to the standard ISO method utilizing the biochemical identification of the isolates [103]. Nevertheless, the enrichment of the food sample as well as the initial _Campylobacter_ load in the matrix significantly affect the isolation frequency and the recovery rate of different subtypes of _C. jejuni_[146] and of many _Campylobacter_  
\begin{table}
\begin{tabular}{l c c c c} \hline \hline
**Characteristic** & _C. jejuni_ & _C. coli_ & _C. lari_ & _C. upsaliensis_ \\ \hline Catalase activity & + 1 & + & + & - 2 or weak \\ Hippurate hydrolysis & + 3 & - & - & - \\ Indoxyl acetate hydrolysis & + & + & - & + \\ \hline \hline \end{tabular} 1
[FOOTNOTE:1]Footnote 1: footnotetext: +: positive reaction. 2
[FOOTNOTE:2]Footnote 2: footnotetext: \(\cdot\): negative reaction. 3  
\end{table}
Table 1: Key biochemical characteristics of _Campylobacter_ species encountered in foods.  
species. In such a case, combining mPCR with the pre-enrichment in Brucella broth and further enrichment in Preston broth of chicken meat samples led to the identification of the majority of isolated amylobacters as _C. coli_ (53%) than _C. jejuni_ (47%) [147].  
The quantitative determination of _Campylobacter_ spp. in contaminated foods and generally in the food chain is crucial, among others in order to comply with imposed EU microbiological criteria for _Campylobacter_ in broiler carcasses [75]. Quantitative molecular PCR-based methods, such as rt-PCR techniques, have been developed for such quantification purposes [148; 149; 150; 151], although currently only the culture-based ISO method is officially approved for pathogen enumeration [105]. Nonetheless, PCR-based methods suffer from the inability to differentiate between live and dead cells of the target microorganism and the apparent PCR signal that may occur from DNA originating from those dead cells. Therefore, quantitative viability rt-PCR assays have been proposed to normalize on the one hand the underestimation of _Campylobacter_ spp. recovered from different food matrices, on the other to allow for reliable differentiation between live and dead _Campylobacter_ and thus accurate estimations of pathogen concentration in foods [79; 152; 153; 154; 155].