A Visualization And Rapid Detection Method Of Adulterated Cow's Milk In Goat Milk Based On Loop-mediated Isothermal Amplification Technology

Dairy products are foods that people often eat in their daily lives,which are rich in nutrients.The core of the quality and safety of dairy products is to control the milk source,and it is of great significance to quickly and accurately detect adulteration of dairy products,which is related to economic interests and safety issues.Compared with cow milk,goat milk is favored by more and more consumers for its higher nutritional value.In order to protect the quality of goat milk and its products,ensure that consumers' rights are not infringed,open the way for the goat milk industry to enter the international market,and at the same time avoid technical barriers to trade,raw materials and products in the production and sales process authenticity detection is an imminent issue.Traditional methods for detecting cow milk adulteration based on proteins and fats have obvious limitations.Because fats and proteins are unstable in food processing(such as high temperature and pressure conditions),thus limiting the widespread use of these methods.The detection methods based on nucleic acid analysis not only have high requirements for operators,but also require professional,expensive and precise instruments and equipment.In order to develop a simple,fast,and economical method for the detection of milk adulteration,in this study,cow milk and goat milk were used as research materials,loop-mediated isothermal amplification(LAMP)was used as the amplification method,DNA was used as the detection object,and SYBR Green I dye was used as the reaction indicator.By optimizing the method of DNA extraction from milk,a new visual loopmediated isothermal amplification method was developed to detect cow milk adulteration.The main content and research results of this study are as follows:(1)Milk was used as the research object to establish a stable kit method of extracting DNA from bovine milk.Based on the classic phenol-chloroform purification method,the key parameters such as incubation temperature,incubation time,sodium lauryl sulfate(SDS)and proteinase K amount were optimized sequentially.The optimal DNA extraction conditions were screened out by measuring the quality of the DNA,and a stable kit method for extracting DNA from bovine milk was established.The method was validated by using deep-processed milk powder and high-and low-temperature sterilized milk.The results showed that the total DNA,mitochondrial DNA(mtDNA)and nuclear DNA(nDNA)extracted from cow milk at different incubation temperature and time were all intact,but the DNA concentration and purity were different.When the incubation temperature was 60?,the incubation time was 10 min,and the amount of SDS and proteinase K were both 50 pL,the quality of the extracted DNA was better,which was the optimal DNA extraction condition.The average DNA mass was 97 ng/kg,and the average purity was 1.44.The kit method established after optimizing parameters was not only applicable to raw cow milk,but also to liquid and solid cow dairy products.Under the optimal DNA extraction conditions,bovine milk somatic cells were lysed fully and thoroughly,and the extracted DNA had high concentration and purity,good integrity and strong PCR amplification performance.(2)Establishment of rapid detection method for cow milk adulterated in goat milk based on LAMP technology.Based on the established milk DNA extraction kit method,the Cytb gene was used as the research object,and two outer primers and two inner primers were used to specifically identify six regions of the Cytb gene sequence.By using SYBR Green I dye,the closed-tube visual detection of the amplification product realized,the reaction result was monitored by visually observaing the color change of the reaction solution,and the amplification product was further subjected to agarose gel electrophoresis to verify the amplification result.The results showed that the reaction solution of the positive amplification samples changed from orange to green,and the reaction solution of the negative amplification samples remained orange,and the visual detection results were easy to distinguish and observe with the naked eye.The established LAMP system can specifically detect the Cytb gene of cow milk,the detection limit was 10 fg/rection,and the detection sensitivity of cow milk in goat milk was 0.01%.Visual detection results were consistent with traditional electrophoresis results,indicating that visual detection could replace traditional electrophoresis detection,and visual detection could avoid aerosol contamination caused by open-lid detection.Compared with traditional PCR methods,the sensitivity and detection limit of the LAMP were both 10 times higher,indicating that LAMP assay was faster and more sensitivity.Using established method to detect cow dairy products in goat milk,0.01%of cow dairy products could be detected.The assay could detect cow milk in goat milk in 45 min with a water bath and it showed great potential in fast detection of cow milk adulteration.