Reverse Transcription–Polymerase Chain Reaction Assay for Species-Specific Detection of Bovine Central Nervous System Tissue in Meat and Meat Products

Abstract

This paper reports the development of a reverse transcription–polymerase chain reaction (RT-PCR) assay coupled with restriction fragment length polymorphism (RFLP) analysis to specifically detect the glial fibrillary acidic protein (GFAP) mRNA of bovine central nervous system (CNS) tissue in minced meat and meat products. RNA extracted from bovine brain tissue and brain tissue from other mammals yielded a 168-bp CNS-specific signal after RT-PCR. The species specificity of the assay can be obtained by subsequent RFLP analysis of the amplified RT-PCR product. To determine the tissue specificity of the RT-PCR assay, various bovine tissues were analyzed. Bovine GFAP mRNA was detected in the brain and spinal cord, and these results are consistent with the reported large amounts of detectable protein in these tissues. Additionally, GFAP mRNA was present in skeletal muscle tissue samples and in some heart muscle tissue samples, but heat treatment of samples prior to extraction resulted in the loss of the non-CNS signals. To evaluate the stability and detectability of bovine GFAP mRNA in comminuted meat and in cooked meat products, mixtures containing bovine brain homogenate at concentrations of 0.5 to 5% were prepared and analyzed. The examination of minced meat with added bovine brain homogenate revealed GFAP mRNA RT-PCR signal stability for at least 7 days at a storage temperature of 4°C. In cooked meat products bovine GFAP mRNA signal was detectable for at least 35 days. Bovine brain homogenate at a concentration of 0.5% was successfully detected in all of the experiments conducted, and no false-negative results were obtained. It is concluded that bovine GFAP mRNA can serve as a sensitive and specific marker for bovine CNS tissue in minced meat and in pasteurized meat products.