Study On LAMP Method For On-site Detection Of Bovine And Goat Derived Components In Milk And Dairy Products

Polymerase chain reaction(PCR)technology has been successfully applied to the identification and detection of animal-derived components in food,but the high instrumentation and technical requirements have limited its application in the field and in primary laboratories with limited resources.Loop-mediated isothermal amplification(LAMP)has similar sensitivity and specificity to PCR,but does not require thermal cycling equipment and is resistant to food sample inhibitors,which is more suitable for the rapid on-site detection of animal-derived components in food.In order to make full use of the advantages of LAMP technology in the field of food testing,this study uses bovine and goat-derived components in milk and dairy products as the detection target.From two aspects of simplified template pretreatment and establishment of closed tube visual detection method,this study established several rapid detection methods suitable for the field.The specific research contents and results are as follows:(1)Direct real-time LAMP for detection of bovine milk components in goat milk.Specific primers of bovine and goat were selected according to mitochondrial conservative sequence,and a direct real-time LAMP technology was established without nucleic acid extraction for the detection of bovine milk adulterated in goat milk.The results showed that the established LAMP method had good specificity,and the detection sensitivity for bovine and goat can reach 0.07pg/μL and 0.7pg/μL,respectively.The direct real-time LAMP technology could be detected 1%milk ingredients in goat milk.Tests on 10 commercial goat dairy products show that the direct real-time LAMP method is consistent with the DNA-extracted real-time LAMP method.The established method avoids the time-consuming nucleic acid extraction process,and can be directly used for LAMP amplification reaction by simple sample processing,which simplifies the sample pretreatment process and is suitable for quick on-site detection of bovine milk components in goat’s milk products.(2)Double LAMP detection of bovine’s milk in goat’s milk.In order to improve the detection efficiency and save time,reagents and samples,this chapter further research and established a dual LAMP detection method based on the difference in melting temperature(Tm)of LAMP amplification products.Through the optimization of primer design,the Tm value of bovine primer amplification product was 80℃ and the Tm value of goat amplification product was 83℃,and the difference in Tm value was used to effectively distinguish between two animal-derived components.This method has good specificity and does not require separate detection of bovine and goat-derived components.It can be quickly detected when one or both of them are present in the system,and there is no cross-reactivity and false positives for other species.The relative value of the adulteration ratio can be achieved by the height of the melting peak,and it is successfully applied to the detection of goat milk and its products on the market.(3)Visual LAMP detection of bovine’s milk in goat’s milk.LAMP technology can quickly identify the amplification products through fluorescent visual colorimetry,but the commonly used organic detection dyes have poor stability and small Stoke’s shift,which makes it difficult to discriminate the fluorescence color of sample from blank.In this study,the ruthenium(Ⅱ)complex[Ru(bpy)2(dppz)]2+with nucleic acid molecule "light-on" effect was exploited as a fluorescent dye to establish a simple,rapid and intuitive visual LAMP method for the detection of bovine milk components in goat milk products.The results showed that the dye[Ru(bpy)2(dppz)]2+was added after the completion of the LAMP amplification reaction,and the presence or absence of the amplification reaction could be determined by observation with a blue light projector.When the target DNA was present,it emitted a strong red fluorescence,which was obviously compared with the excited blue light,and it was easier to distinguish visually.This method has good specificity and sensitivity,and the adulteration in goat milk could be detected 0.1%milk.(4)Closed tube visual LAMP detection of bovine’s milk in goat’s milk.Due to the high sensitivity of LAMP reaction,adding[Ru(bpy)2(dppz)]2+dye to the open cap after the reaction can easily cause aerosol pollution.In order to avoid contamination and inhibition of LAMP amplification reaction by directly adding high-concentration dyes,a simple tube separator was further designed and optimized.Both the LAMP amplification reagent and the detection dye were added before the reaction but they were not in contact with each other.After the amplification reaction was completed,they were mixed to produce a fluorescence signal,which not only realized highly sensitive fluorescence detection but also avoided the aerosol pollution caused by opening the cap.By optimizing the amplification detection conditions,adulterated 0.1%bovine’s milk in goat’s milk could be detected within 40 minutes.This method has the advantages of fast,intuitive,low cost and little pollution risk,and can provide technical support for rapid on-site detection of authenticity of goat milk.