Study On The Application Of Sea Method In Pork Adulteration Detection And Potential-assistant Pretreatment

Due to its high protein content and low fat content,beef and mutton taste delicious and nutritious.However,due to the high feeding cost of cattle and sheep,the price of beef and mutton is relatively high.Pork is similar in color and texture to mutton or beef,and is widely used as alternatives for these two kinds of meat.The addition of pork meat in beef or mutton without declaration is fraudulent activity,which may greatly threaten ethnical options or religious practices of consumers,for example Muslims.The improvement of living standard for people in China and the modernization of China's food industry have resulted in the increase of demand and production of animal food production.At the same time,for pursuing higher economic profit,food manufactures might replace expensive mutton or beef with cheap meat,such as edible pork,chicken,duck,horse and inedible rat meat,fox meat,etc.With the resolution of the adulteration of meat products more and more prominent in the world,the detection of adulteration of meat products on site with simple equipment is of more and more important significance for food safety,religious belief and other fields.Many techniques have been developed to identify pork sources in other meats,such as morphology,spectroscopy,chemical techniques and protein analysis.The accuracy of these methods is based on a certain degree of experience and skill,and is affected by complex food simulations,so it is often difficult to detect pork from several meat categories.Due to the higher stability of DNA molecules,nucleic acid-based molecular technology is more advantageous for the detection of adulteration of meat products,and has the characteristics of fast,high sensitivity and specificity.However,the thermal cycling process in PCR requires a high level of laboratory equipment,and the reproducibility of PCR-RFLP is widely questioned due to the long processing time.Although conventional nucleic acidamplification methods can be tested,they require complex equipment,skilled technicians,and long operating times,which limit their field testing.In this study,a simple denature-bubble mediated Strand Exchange Amplification(Strand Exchange Amplification,SEA)with only a pair of primers and a polymerase was used to identify the adulteration of pork by targeting the species-specific mitochondrial DNA sequence.The SEA method showed good specificity for pork detection,in addition to the porcine endogenous components can not cause amplification,and could detect pork DNA as low as 30 pg/?L.In binary mixtures,SEA method can detect as low as 1% of total pork DNA by coloration and fluorescence,in most countries,unintentional levels of pollutants are allowed the label pollution that can be exempted is 1.0-3.0%,thus SEA method meets the requirements of adult meat adulteration detection.In this study,combined with the rapid tissue DNA extraction method,the whole detection process can be completed within 1 hour,and only a simple metal bath or water bath can realize the entire detection process,and can output visual detection signals.Compared with the industry standard PCR method in the field of nucleic acid detection,SEA method has simpler equipment requirements and faster detection time with the same specificity and sensitivity.Therefore,the method of this study has the advantages of simplicity,rapidity and specificity for the field identification of porcine derived components.In the research process,it was found that the concentration and quality of meat DNA extracted during the previous meat product processing had a crucial impact on the detection time and detection accuracy of subsequent amplification reactions.Therefore,this study described the preliminary investigation results of electric-field assisted hybridization for the extraction and enrichment of nucleic acid DNA.Electrochemical assisted hybridization accelerated the process of DNA molecular hybridization,realized rapid specific nucleic acid extraction,and further optimized the detection process of adulteration of meat products by using the movement law of DNA molecule under the influence of electric field.