Study On Detection Of Adulteration In Beef And Mutton By Multiplex Real-Time Fluorescent PCR

China’s meat production accounts for about one-third of the world’s total production,and sufficient supply of meat resources provides favorable conditions for the development of China’s meat industry.At present,the consumption demand for meat and meat products among Chinese residents is upgrading,shifting rapidly from quantity to quality,and from basic necessities to nutrition and health.As a source of high-quality protein for human nutrition,the safety of meat and meat products is closely related to people’s physical health.However,adulteration in meat products still occurs from time to time,and adulteration identification and traceability analysis have become hot issues in monitoring.The mixing of meat products of different animal origins not only disrupts the sales market order,infringes on consumer rights,but also lays hidden dangers for the spread of potential animal diseases.There is an urgent need for a fast and accurate detection method.In this study,animal mitochondrial COX I gene was used to screen species specific DNA sequences by comparing genes,and multiple PCR technology was used to explore and establish qualitative and quantitative detection methods for six animal derived components in meat and meat products,so as to shorten detection time and improve detection efficiency.To provide technical support to meet the urgent needs of regulatory authorities for rapid identification of animal derived ingredients.Four extraction methods were compared,namely improved CTAB method,improved SDS method,non centrifuge column method(reagent kit),and centrifuge column method(reagent kit).Raw meat from six animal derived ingredients and cooked meat from three processing methods were selected as samples,and the non centrifuge column method based on protease K with the highest extraction rate was selected as the DNA extraction method for this study.Optimization was carried out to improve the efficiency of DNA extraction and provide technical support for subsequent PCR experiments.This experiment targeted the mitochondrial COX Ⅰ gene of cattle,sheep,chickens,ducks,horses,and pigs.Six pairs of special primers and probes were designed to optimize the primer concentration and annealing temperature,and the specificity,sensitivity and repeatability of the method were validated.The results suggest that the fluorescence PCR detection method with six pairs of single primer TaqMan probes had good specificity,and the minimum detection limit of DNA mass concentration for cow,sheep,chicken,duck,horse,and pork components were respectively,10-4 ng/μL,10-3 ng/μL,10-4 ng/μL,10-4 ng/μL,10-3 ng/μL,10-4 ng/μL.It has good sensitivity and high repeatability.A multiplex fluorescence PCR detection method was established based on a single TaqMan probe,with an amplification program of 95℃ for 600s,95℃ for 15s,60℃for 30s,and 40 cycles.It can simultaneously detect 6 animal derived components with a detection limit of 0.1%(w/w)and a detection time of 90 minutes.A new method was applied to detect the adulteration of 154 beef and mutton samples,and it was found that 20.78%of the results did not match the label.There is still a situation in the market where low-priced meat is used to partially or completely replace high-priced meat.Overall,the adulteration rate of beef and mutton products in the agricultural market is higher than that of commercial supermarket samples,and the adulteration rate of mutton products is higher than that of beef products.