Real-time PCR Method For The Detection Of Adulteration In Dairy Products

Objective: To establish real-time PCR protocols for detecting principalcomponents of bovine, ovine and frequent adulterated ingredients of cereal, rice andsoybean in dairy products. Meanwhile, explore quantification detection system tomeasure the concentration of principal components in milk powder based on thequalitative detecting systems.Methods:1.Qualitative system for the detection of adulteration in dairy products.Specific primers and probes, targeting on bovine mitochondrial cytochrome b (cyt b)gene、ovine mitochondrial12SrRNA gene、the homologous region in a conservedregion of the rbcL gene of rice, maize, wheat, sorghum and barley、rice root-specificgene gos9gene and lectin gene of soybean, were designed to established the real-timePCR system for detection of main and possible adulterated ingredients in dairyproducts, which were evaluated by specificity and sensitivity tests. The specificitytests were conducted with target species and non-target species DNA, while thesensitivity tests were conducted using ten-fold DNA serial dilutions of each targetspecies and the different concentration of simulation milk powder samples. Establishedreal-time PCR system were further applied to the detect market dairy samples to verifythe application ability of the methods.2. Quantitative system for measure the concentration of principal components inmilk powder. Based on qualitative detection real-time PCR system for bovine, thequantitative curve between different concentrations of bovine milk powder and theirCycle threshold(Ct) targeted in bovine mitochondrial cytochrome b (cytb) gene wasestablished The credibility of the quantitative system was assessed by the recoveryexperiments and its application ability were further verified by being applied to themeasure of the principal components in market milk powder. Results:1.Qualitative detection of adulteration in dairy products.(1)The specificmethods for bovine could only detect DNA fragments of bovine and the specificmethod for ovine could only detect DNA fragments of ovine within35cycles with nocross reaction to other non-target species. The detection threshold were0.1ng、0.01ngfor target DNA and1%、0.5%(w/w) for the simulation samples. The assays whenapplied to dairy samples collected from the retail trade has confirmed the speciesindicated in the labels.(2)The universal primers-probe system for cereal(Ct>35) and thespecific primers-probe assays for rice and soybean(Ct>40) were all proved to be highlyspecific for target species with no cross-reaction to other non-target species. Thedetection limit was0.01ng、0.1ng、0.1ng for pure DNA and0.5%、0.1%、0.5%(w/w)foreach target species spiked in dairy mixtures. With the established systems applied tothe detection of commercial dairy samples, all samples except one yield correspondedresults which showed identical to the species indicated in the food labels.2. The standard curve equation for quantifying bovine in milk powder wasy=-0.057x+32.759.The coefficient of correlation R were0.964. Recovery rates werebetween109%~134%.Three bovine milk powder samples in eight were detectedcontaining below50%principal component,and one of the three samples was provedto be adulterated cereal ingredients other than rice.Conclusions:The qualitative real-time PCR protocols for detecting principal components ofbovine, ovine and frequent adulterated ingredients of cereal, rice and soybean in dairyproducts were universal, specific, sensitive and feasible, which were capable to findadulteration in dairy products. Quantification systems for bovine could identify thecontent of the principal components of bovine-derived ingredients in milk powder,which could be used as the detection methods for identifying the adulteration of milkpowder.