In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan mgb and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 10 2 CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.Mycobacterium avium subsp. paratuberculosis is the causative agent of ruminant paratuberculosis (Johne's disease), which has become a worldwide problem. There is controversy regarding its zoonotic capacity and potential role in the human Crohn's disease (14). Because of these reasons, a rapid, costeffective, and automated diagnosis of this pathogen is a high priority task not only for animal breeders but also for the food production industry and for public health institutions. Culturebased detection of M. avium subsp. paratuberculosis is timeconsuming, labor-intensive, and therefore not suitable. The PCR has been shown to be a powerful tool in microbiological diagnostics (12,43). Guidelines for diagnostic quality assurance have been set by the International Organization for Standardization (7,8). Standardized PCR and real-time PCR methods should fulfill numerous criteria, including a high detection probability with regard to the investigated matrix, the sample preparation, and DNA extraction as well as high specificity, robustness, and user-friendly protocols. In this context the real-time PCR technology offers the possibility for a onestep and closed-tube reaction (13).As a molecular reference marker for the confirm...

The emergence of a new variant of Creutzfeldt-Jacob disease during the bovine spongiform encephalopathy epidemic has focused attention on the use of tissues from the central nervous system (CNS) in food. For efficient consumer protection, European legislation prohibits several bovine tissues, encompassing mainly the central nervous system, from the food chain. A quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was designed to identify bovine spongiform encephalopathy risk material in meat and meat products. This was based on an mRNA assay that used bovine, ovine, and caprine glial fibrillary acidic protein (GFAP) encoding gene sequences as a marker. The real-time RT-PCR assay allowed the detection of bovine, ovine, or caprine CNS tissues in meat and meat products. Bovine brain at a concentration of 0.01% yielded a positive PCR reaction. The real-time RT-PCR assay included a housekeeping gene as an endogenous control. The detection was not affected by heat treatment of the meat products. The quantitative real-time RT-PCR detection of GFAP mRNA appeared to be useful as a routine diagnostic test for the detection of illegal use of CNS tissues in meat and meat products. The stability of the specific region of GFAP mRNA also allows the detection of CNS tissues after meat processing steps. The use of organ- and species-specific subunits of mRNA might be a promising approach for the detection of other banned tissues.

The removal of central nervous system (CNS) tissues as part of bovine spongiform encephalopathy (BSE) risk material is one of the highest priority tasks to avoid contamination of the human food chain with BSE. No currently available method enables the real-time detection of possible CNS tissue contamination on carcasses during slaughter. The fluorescent pigment lipofuscin is a heterogeneous, high-molecular weight material that has been shown to be enriched in high concentrations in neuronal tissues. In this study, lipofuscin fluorescence was investigated as a marker for real-time detection of CNS contamination. Front-faced fluorescence spectra of brain and spinal cord samples from 11 cattle gave identical, reproducible fluorescence signal patterns with high intensities. The specificity of these spectra was assessed by investigating 13 different non-CNS tissues enabling the differentiation of brain and spinal cord by signal intensity and structure of the spectra, respectively. Small quantities of bovine spinal cord were reliably detected in the presence of raw bovine skeletal muscle, fat, and vertebrae. The presented data are a fundamental basis for the development of a prototype device allowing real-time monitoring of CNS tissue contamination on bovine carcasses and meat cuts.