Research On Detection Technology Of Adulterated Meat Based On Cross-primer Isothermal Amplification

Meat adulteration occurs recently from time to time due to that some traders intentionally searching for illegal benefits,which seriously hurt the interests of consumers and may caused sensitive issues such as ethics or religion.To provide technical support for security of meat marketing,the objective of this study is to establish a rapid and reliable identification method of meat authenticity with combination of the cross-primer isothermal amplification(CPA)and colloidal gold nucleic acid test strip.In addition,a rapid DNA extraction technique was developed and a closed detection device for amplification and visual testing was designed.The main findings were shown as follows:1.To meet the requirement of isothermal amplification,some elements such as reagents,equipment,and operations steps were simplified and optimized from the conventional animal DNA extraction procdure.The optimization included the increase in sampling volume of meat sample from 0.1 g to 5.0 g,the removement of one step and one reagent from the conventional,while improvement of the representing aspact of meat samples,saving reagents and shortening of time consume by 30 mins.The evaluation by sheep/fox samples showed that the detection limit was 9%.2.A simple detection device integrated isothermal amplification and colloidal gold nucleic acid test strips was designed,which was easy to operate and small in size.The results could be observed visibly by inversion,reducing the percentage of the false positive cases caused by aerosol contamination.The sheep-specific CPA detection system combined with electrophoresis test device was effective,and the contamination rate was reduced by 19%compared to the air-exposed test strip.Combined with rapid DNA extraction technology,it could achieve the application of CPA in the rapid detection of meat source components.3.A CPA primer system based on fox-specific DNA segments was designed and optimized,and an effective detection technology with combination of CPA amplification system and colloidal gold nucleic acid test strips,which could quickly distinguish fox meat from donkey,sheep,pigs,cattle,duck and rat meat with a sensitivity of 10 ng·?L^-1,and the detection limit of 1%for fox components from sheep and fox mixed meat samples by using the new DNA extraction method and the closed device.4.Using a set of universal primer systems combined with specific additional labeled primers and clever conversion of CPA primer labeling methods,a CPA method for authentication of multiple species components combined with colloidal gold nucleic acid test strip was established.The feasibility and optimization of the method were verified by the CPA universal primer system testing of donkey,sheep and fox meat,and further confirmed by the identification experiments of four kinds of meat including cow,sheep,fox and pig with the sensitivity as 0.1 ng·?L^-1 and the detection limit of 1%,indicating DNA from multiple meats could be specifically detected.Moreover,the simultaneous detection of two kinds of meat as pig and fox was achieved,with a double detection line of colloidal gold nucleic acid test strip.