Novel approach for detecting prohibited species-specific central nervous system tissue contamination in meat by one-step real-time reverse transcriptase PCR.

The dissemination of prohibited species-specific central nervous system (CNS) tissue contamination in meat must be tracked to mitigate human health risk associated with bovine spongiform encephalopathy. The efficiency of compliance monitoring and risk control measures taken by concerned regulatory authorities at meat production facilities to avoid such contamination depends on the ability to detect CNS tissue with a reliable and adequately sensitive quantitative method. A rapid and convenient one-step real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was developed based on the absolute quantification of glial fibrillary acidic protein (GFAP) mRNA as a marker for CNS tissue contamination in meat. The GFAP RNA quantity corresponding to a percentage of CNS tissue in artificially spiked meat was determined using an appropriate in vitro transcribed target GFAP RNA as a calibration standard in the assay. The assay had a linear dynamic range of 10(2) to 10(9) copies of target RNA and was able to detect 0.01% CNS contamination in meat. Further evaluation consisted of an analysis of 272 random meat cuts from carcasses and 109 ground meat samples received from a federally inspected abattoir and two meat processing facilities, respectively, over a 5-month period. The analyzed samples were all negative for CNS tissue contamination at an arbitrarily set lower threshold of 0.025%. Overall, the newly developed one-step qRT-PCR may be useful as an objective quantitative compliance monitoring tool and for setting an acceptable low tolerance threshold for such contamination in meat.