Application Of Loop-mediated Isothermal Amplification In Detection Of Adulterated Meat

The meat adulteration was a severe food fraud,the manufacturer mixed other species meat in high-price meat or meat product in some way,it was difficult to distinguish the fraud via appearance,and the aim of improving profit was achieved.The effect of meat adulteration had been greatly unfavorable for development of our national meat industry,the detection methods for meat fraud was urgently needed for market surveillance,the current methods for detecting meat adulteration were based on morphology,metabology,proteomics and genetics,among which the commonest methods were established upon genetics.Loop-mediated Isothermal Amplification?LAMP?was a nucleic acid amplification technology with advantages of high sensitivity,specificity and so on,and the technology had been widely used in agriculture,medicine,botany,food science and other fields due to its prominent advantages.The objective of this study was to establish the LAMP methods for detecting meat fraud.The specific research work followed as below.1.The visual LAMP system for detecting pork component had been established with 4-?2-pyridylazo?resorcinol monosodium salt as indicator,the LAMP primers were to be designed with cytochrome b gene of pork as target,the reaction temperature optimized,the sensitivity and specificity determined.The results indicated that,the optimal temperature was 57oC,the absolute sensitivity was 10 pg,the relative sensitivity was 1%,the established method was of high specificity.Moreover,the pretreatment of DNA extraction had been simplified,and the total detection time had been sharply shorted.2.The visual LAMP system for detecting donkey component had been established with 4-?2-pyridylazo?resorcinol monosodium salt as indicator,the LAMP primers were to be designed with cytochrome b gene of donkey as target,the reaction temperature optimized,the sensitivity and specificity determined.The results indicated that,the optimal temperature was 59oC,the absolute sensitivity was 1 pg,the established method was of high specificity,which can effectively distinguish horse meat with donkey meat.3.The Real-time LAMP system for detecting rabbit component had been established with SYBR GREEN I as indicator,the LAMP primers were to be designed with cytochrome b gene of rabbit as target,the reaction temperature and Mg²⁺optimized,the sensitivity and specificity determined.The results indicated that,the optimal temperature was 63oC,the absolute sensitivity was 8 pg,the relative sensitivity was 1%,the established method was of high specificity,and the detection can be finished within 20 min.