Establish and Application of a Real-Time Fluorescence-Based Polymerase Chain Reaction Assay for Detection of Bovine-Derived Materials in Meat Products
MIAO Li1, LI Zhijuan2, WANG Shan2, ZHANG Xiuping2, CHEN Jing2
1.Central Quarantine Laboratory of Henan Entry-Exit Inspection and Quarantine Bureau, Zhengzhou 450003, China;
2.Identification Consulting Center of Henan Inspection and Quarantine, Zhengzhou 450003, China
Abstract：A real-time fluorescence-based polymerase chain reaction (PCR) assay that enables rapid and accurate identification of adulterated beef products was developed using TaqMan probe for the rapid and effective detection of bovine-derived materials in meat products. A pair of specific primers and TaqMan probe was designed according to the conserved sequence of the bovine β-actin gene. PCR reaction conditions were optimized using pMD-18T-96-beef plasmid as standard. The results showed that the pMD-18T-96-beef plasmid was confirmed to be valid by PCR, sequencing and enzyme digestion and the developed standard curve produced a good linear relationship between Ct value and initial amounts of total DNA with correlation coefficient and slope of 0.98 and −3.345, respectively. The sensitivity was 46.1 copies/μL. No cross-reactions were found in specimens containing pork, mutton, chicken, duck and goose. The intra- and inter-assay variable coefficients were less than 0.98% and 0.33%, respectively, suggesting good repeatability. A total of 40 beef product samples from the market were measured by this method and the routine real-time PCR method from the industry standard SN/T 2051—2008. Of the 40 samples, 3 were detected as positive as consistently indicated by the two assays. This real-time PCR method with strong specificity and high sensitivity could provide a useful approach for the identification of meat adulteration.
