Establishment of Multiplex Real-time fluorescence PCR for Detection of Mink (Mustela lutreola), Pig (Sus scrofa) and Rat (Mus musculus) Derived Components in Beef and Mutton
Abstract：Abstract At present, events about meat adulteration were common occurred, for example, mix fox flesh to cooked donkey flesh in Wal-Mart and horsemeat event spreading EU countries. Now, the method of meat adulteration has been adulterated by one ingredient to a mixture of ingredients. This kind of fraud disturbs the market order seriously. Countries have paid more and more attention to meat adulteration, the identification and traceability of meat adulteration analysis become a hot topic on the world. Traditional meat identification was authenticate the meat appearance, color, odour, falvor and hardness by vision, touch, smell and taste, these methods cannot meet the needs of meat quality and source authentication now. In recent years, with the development of modern analytical instrumentation and biotechnology, identification techniques for meat adulteration have also been well developed. Including protein-based detection methods, enzyme-linked immunosorbent assay (ELISA) methods, chromatography analysis methods and DNA-based detection methods. Protein-based detection methods can detect quantitatively, but it is not suit for animal origin analysis. Many ELISA kits have already been commercially developed, but their application is limited due to protein activity. DNA-based detection methods have the characteristic of simple, short detection time and accurate, of them, fluorescence PCR detection method has the advantage of high sensitivity, high specificity and high throughput, it will become the future direction of the meat source identification. Multiple fluorescence qPCR was used in this experiment. In order to identify the 3 ingredients of mink source, porcine source and rat source in mutton and beef, a pair of universal primers were designed based on the mitochondrial 16S rDNA conserved region of the 3 different animals and 3 kinds of species-specific molecular beacon probes with strong specificity were designed based on the mitochondrial 16S rDNA of mustela vison, sus scrofa and mus musculus. The specific verification of the 3 probes, the sensitivity verification of the experimental system and the test of the detection limit of the samples were carried out respectively in this experiment. In the sensitivity verification experiment, the 3 probes can effectively eliminate the interference of DNA from other species and specifically amplified in both monoplex fluorescence qPCR and multiplex fluorescence qPCR systems. In the sensitivity verification of experiment, seven dilution gradients of DNA concentration were set up and 6 parallel samples for each gradient, it was determined that the sensitivity of the system was 0.01 ng/μL. In the test of the detection limit of samples, 3 kind of doping methods were set up, 7 different dopant mass ratios were set up in each doping method and 4 parallel samples for each dopant mass ratio, the sensitivity of the test was determined to be 1% (mass ratio). This method provides a scientific basis for the origin identification of multi species, puts forward a new way to control the authenticity of animal raw material sources, and provides powerful technical reference for enterprise supervision to rectify the mess of meat industry.
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