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The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (C T ) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R 2 ) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 g/ml) and PMA (51.10 g/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10 4 ) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.

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A total of 267 cysts were collected from March to December 2004 from two main abattoirs in northern Germany. The cysts were classified by the usual organoleptic methods during meat inspection as Cysticercus bovis. The reported prevalence of cysticercosis in the abattoirs was 0.48 and 1.08%, respectively. The cysts were examined macroscopically for description of their morphology and constituents and classified as viable or degenerating (dead). The DNA was extracted from these cysts and subjected to polymerase chain reaction (PCR) for evaluation of the detection methods used and to make certain that the cysts did indeed belong to C. bovis, as indicated at the slaughterhouses. Two sets of primers were used with different sensitivity levels. The first, HDP1, was able to detect 200 fg of Taenia saginata DNA and 100 pg of C. bovis DNA. The other primer set, HDP2, was able to detect 1 pg of T. saginata DNA and 1 ng of C. bovis DNA. No more than 52.4% of the samples tested positive for C. bovis in the PCR using both primers, while 20% of the viable cysts and 49.2% of the degenerating cysts tested negative with both primers.

Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes.

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