Research On Rapid Identification Of Meat-derived Components Based On Constant Temperature Microfluidic Technology

"Food is the heaven for the people,and safety is the first for food." Food safety is as important as Mount Tai.As the main source of protein,meat products occupy a large proportion of production and consumption in food.In order to seek illegitimate interests,some illegal vendors have adulterated animal-derived ingredients during the processing of meat products.This kind of food adulteration has not only hidden safety hazards,but also greatly harmed the interests of consumers and disrupted market order.Healthy development has created potential conflicts with ethnic and religious beliefs,and has also created barriers to the import and export trade of related products in our country.Therefore,the General Administration of Quality Supervision,Inspection and Quarantine issued the "Warning Notice on Strengthening the Inspection and Quarantine Supervision of Imported and Exported Meat and Meat-Containing Products",which requires testing of imported meat and meat-containing products for adulteration and other ingredients.Animal-derived ingredients in food The establishment and application of detection methods are imminent,especially the rapid detection technology suitable for large-scale screening and detection at the grassroots level.The traditional detection methods are cumbersome to operate and take a long time.Multiplex PCR,real-time quantitative fluorescent PCR,digital PCR and other expensive instruments that rely on temperature rise and fall are not conducive to actual on-site detection and law enforcement.Loop-mediated isothermal Amplification(LAMP)is an ideal method created on the basis of PCR,which can well solve the shortcomings of current genetic testing.In this study,loop-mediated isothermal amplification(LAMP)rapid detection technology was used,and specific primers were designed according to the mitochondrial gene sequences of cattle,buffalo,sheep,goat,mice,and rats to specifically identify six target sequences.In an independent area,the LAMP reaction system(primer concentration,Bst polymerase addition amount,reaction temperature)was optimized,and the sensitivity,specificity and stability of the primers were evaluated,and the LAMP detection method of six animal-derived components was successfully established.Combining the simplicity of constant temperature amplification of cattle and sheep with the high-throughput characteristics of microfluidic chips,a constant temperature microfluidic detection technology has been developed to verify specific experiments,5%,1%,0.5%,0.1% Sensitivity experiments with different mass blending ratios and the detection of actual samples can further realize the rapid detection of animal-derived ingredients in food,and realize the rapid,high-throughput,and automated detection of animal-derived adulterated ingredients in meat products.The results of the study are as follows:(1)The animal-derived LAMP detection system established in this study separately tested yellow cattle(Gen Bank No.MF925711.1),buffalo(Gen Bank No.MT182644.1),goat(Gen Bank No.LS992662.1),and sheep(Gen Bank No.MT182644.1).MT768241.1),mice(Gen Bank No.KY018919.1),and rats(Gen Bank No.KP244683.1)were developed,and the primer concentration that affects the reaction system,the amount of Bst polymerase added and the reaction temperature were optimized,and According to the peak time,peak height,repeatability and amplification efficiency and other indicators to confirm the final reaction system and reaction conditions.The minimum detection limit of scalpers is 0.1 fg/(?)L,and it can complete the detection of 0.1% of artificially adulterated scalpers;the minimum detection limit of buffalo is 1 fg/(?)L,and the minimum detection limit of artificially adulterated buffalo samples is 0.1%;Sheep showed good specificity under optimal reaction conditions,and the minimum detection limit of sheep was 1 fg/(?)L;goat LAMP system could specifically detect positive samples,and the minimum detection limit of goat was 0.1 fg/(?)L Both goat and sheep LAMP detection methods can detect artificial adulterated samples with a content of 0.1% of the corresponding source component;the minimum detection limit for mice and rats is 1 fg/(?)L,and it can be used for corresponding animals An artificial adulterated sample with a source ingredient content of0.1% was tested.The bovine,ovine,and murine-derived LAMP detection method established in this study has the advantages of high sensitivity,strong specificity,good stability,simple operation,and high cost performance.It supplements and perfects the molecular biology detection method of animal-derived detection,which is suitable for basic-level detection units or on-site rapid detection.It has strong practicability and has good research value,promotion value and application prospects.2.This study combines the loop-mediated isothermal amplification method with the microfluidic chip technology to establish a constant temperature microfluidic detection method for buffalo and goats.The nucleic acid is extracted from the sample and added to the chip,and the constant temperature reaction is performed in the instrument.After 30 minutes of reaction It can detect samples with 0.1% bovine-derived content and 0.5% sheep-derived samples with good specificity.Compared with the national standard PCR method,the thermostatic microfluidic chip detection method has the same detection rate.It can meet the needs of actual sample testing.The research and development of constant temperature microfluidic detection technology can realize the detection of multiple animal-derived components at one time.It has the advantages of reduced reagent consumption,constant temperature amplification,short reaction time,low requirements for detection equipment,and automated analysis.It can be used in regulatory agencies Daily supervision and inspection can also be used for internal self-inspection of enterprises to control the quality of products.The results of this research are of great significance for ensuring the safety of people's lives,safeguarding consumer interest,promoting the healthy development of related industries,and avoiding ethnic conflicts and disputes caused by food problems.