Application Of Screen-printed Electrode Biosensor Combined With MT COI Gene In Meat Identification Technology

Safety and quality of meat products have continued to be concerned by the entire society.Nucleic acid-based analysis method is commonly used for meat identification and other inspection and quarantine work.DNA barcoding is one of the key developments in modern biotechnology,and is widely used in species identification as a powerful complement in traditional classification methods.Mitochondrial cytochrome C oxidase subunit I(COI)gene has been determined as a standard DNA barcode nucleic acid sequence in research for the animal classification and identification.However,independent DNA barcoding has disadvantages.By combined with biosensors,the commercial expansion of barcode technology can be effectively solved.But there has been little research in technology combination between the biosensor and DNA barcode gene.Therefore,this topic is devoted to search for building a platform based on combining with mitochondrial COI gene(as recognition component)and screen-printing biosensor.Through the investigation of four kinds of raw meat,the adulteration model of porcine-bovine mixture and the commercial samples,the specificity,sensitivity and commercial applicability of the biosensor platform was evaluated,moreover,the practical value in meat fraud supervision system was explored.The main work is carried out as follows:1.It showed the optimal conditions for the electrochemical biosensor:the optimal binding time of the probe to the target sequence is 45 minutes;the optimal hybridization temperature is 45 minutes;the optimal working concentration of methylene blue is 1.5×10-5 M;the optimal incubation time is 15 minutes.A great linear relationship between the raw beef and raw pork target sequences in the range of 10-13-10-5 mol/L devoted the lowest detection limits respectively with 5.048×10-14 M in beef and 3.491×10-14 M in pork.It is preliminarily verified that the electrochemical biosensor constructed is feasible.2.The experiment was designed to simulate the adulteration of porcine/bovine mixture,and explore the specificity,sensitivity and practicability of the COI gene screen-printed biosensing platform.When the adulteration ratio of the mixture is more than 3%,the sensing platform showed outstanding recognition ability;with modified probe,when the mixed raw meat amount in 50 ng,the electric signal value of the reactive dye methylene blue is more stable.When the ratio is less than 25%or greater than 25%,the mixed binary can be identified.It was confirmed that the specific probe can complete the identification of the raw porcine/bovine mixture adulteration model and the commercially available meat sample on the electrochemical biosensor.3.Quantitative analysis of the porcine/bovine mixtures using real-time fluorescent PCR.Compared with the relevant factors and conclusions in Work 2,the practical application of the COI gene screen-printed biosensing platform constructed in this experiment has been prospected.