Establishment And Application Of Multiplex QPCR And MNP Marker High-throughput Detection Methods For Animal-derived Components In Meat Products

At present,most of the meat adulteration identification methods are suitable for the identification of single animal-derived component,and single-source detection methods are cumbersome to operate,time-consuming and laborious,and can’t meet the needs of high-throughput detection.So the establishment of accurate and efficient detection methods for animal-derived ingredients is significant to maintain a good environment for food safety.In this study,a multiplex real-time quantitative PCR（qPCR）method for the simultaneous detection of four animal-derived components and a multiple nucleotide polymorphism（MNP）marker method for the simultaneous detection of ten animal-derived components based on the principle of high-throughput sequencing were established,and the main results were as follows:1.Based on the cytb gene of sheep,duck,chicken,and D-loop gene of pig,primer probes were designed,the annealing temperature and primer-probe concentration were optimized,and a multiplex qPCR method that could detect duck,pig,sheep,and chicken-derived at the same time was established.The results showed that the absolute sensitivities of duck,pig,sheep,and chicken-derived were 1×10^-4 ng/μL,1×10^-4 ng/μL,2×10^-4 ng/μL,and 2×10^-3 ng/μL,respectively.In simulated mixed sample testing,the minimum detection limit was 0.01%（w/w）.This method had good specificity and could realize the quantitative detection of four animal-derived components at the same time.2.70 pairs of primers were designed with the chromosomal genes of 10 derived components of goat,sheep,cattle,yak,horse,donkey,chicken,duck,goose,and pig,high-throughput sequencing was performed after multiplex PCR amplification to establish an MNP marker method that could simultaneously identify multiple loci of 10target components.The results showed that in a single sample,MNP marker method could specifically detect species sites and accurately identify species origin,and the number of sites detected was as follows:9 of horse and goose,8 of chicken and donkey,7 of goat,5 of duck and pig,4 of cattle,3 of sheep and 2 of yak.In simulated adulteration mixed samples,a minimum of 0.1%donkey-derived components could be detected;The accuracy of this method was 70%～100%and high sensitivity.3.To explore the applicability and detection limits of the two methods in meat sample detection in different processing methods.The results showed that for the simulated mixed meat samples of dry,boiled,and high-temperature and high-pressure steaming,the detection limits of the MNP marker method were respectively 1%,0.1%,and 1%（w/w）,and the detection limits of multiplex qPCR method reached 0.01%（w/w）,the detection limit of the two methods was lower than or equal to 1%（w/w）recommended in GB/T 38164-2019.4.The two established methods were applied to the detection of 17 batches of commercially available samples to verify the practical feasibility of the multiplex qPCR method and MNP marker method,and the results showed that 7 batches of samples were detected by both methods that didn’t match the label identification components,including 2 batches of mutton products,3 batches of beef products,1 batch of donkey meat product and 1 batch of pork product,compared with the standard detection method,the detection throughput and accuracy of multiplex qPCR method and MNP marker method were improved,indicating that the established method could be used for the detection of actual samples.