| Research background and purpose:Aquatic product safety is an important factor affecting food safety.The residue of malachite green(MG)is one of the most representative fishery problems.In the process of aquaculture,MG is illegally used and remains in the fish body;fish can build up in the body when consumed by humans,causing life-threatening health problems.Comprehensive testing of MG use in aquatic products is one of the ways to effectively improve the quality and safety level of aquatic products and plays an important role in creating a good aquaculture environment and improving the quality and safety guarantee system.At present,HPLC and LC-MS are used for MG detection in domestic industry quality supervision departments.This kind of method has high sensitivity and few false positives,but the instrument is expensive and requires a long analysis time,operation threshold,and poor timeliness,which makes it impossible to carry out rapid field detection.In this thesis,the rapid detection method of MG based on aptamer recognition colloidal gold flow chromatography strips was discussed,hoping to provide help for the rapid detection of fishery drug residues.In this thesis,aptamers are used instead of antigens and antibodies as recognition elements to explore the application and value of aptamers in MG recognition.The aim is to construct a simple,fast and low-cost visual detection method,provide a new idea for the rapid detection of aptamer recognition technology in the MG field,and supplement the research gap.Materials and methods:1.A colorimetric method was used to verify the feasibility of the selected sequence and simply predict the reaction ratio between the sequence and the sample.Colloidal gold was prepared by the sodium citrate reduction method and characterized by ultraviolet visible spectrum scanning.According to the characteristics of colloidal gold discolouration precipitation in salt,the protective effect of the aptamer on colloidal gold particles was verified,and the identification results of the reaction with the target substance and aptamer were verified.Moreover,conditions such as KCl concentration,aptamer coating amount and p H value were simply optimized,and methodological verification was conducted to prove the feasibility of the method.2.Preparation,optimization and detection of colloidal gold strips.The mercaptogenated MG aptamer was used to conjugate colloidal gold to obtain a gold aptamer,and the coupling ratio of colloidal gold and aptamer and the amount of gold aptamer were optimized.The optimization of influencing factors of the test strip device on the colloidal gold test strip was studied,and the optimization of card reading time,incubation time,amount of T/C nucleic acid sequence,amount of Au NPs-Apt addition,buffer composition and other conditions were investigated.To determine the detection limit and detection range of the strip,the repeatability and specificity of the strip products were evaluated.The recovery rate was detected in the actual water sample.Results:1.A visual detection method for MG was established based on colloidal gold technologyandaptamers.Theobtainedaptamersequence5’-AGGGTTAGGGTTAGGGTTAGGG-3’was combined with colloidal gold to construct a colloidal gold-aptamer colouration sensor.Under the optimal experimental conditions,the linear relationship between MG and A620/A520 was good in the concentration range of 0.1-10μg/m L.The R~2value was 0.9701,and the linear regression equation was y=0.189*log Conc.MG+0.5845,and the detection limit was0.032μg/m L.The recoveries of MG were 86.3%-94.62%,and the RSDs were3.67%-8.34%.2.A colloidal gold test strip based on aptamer recognition was established for the detection of MG in water.Under the optimal experimental conditions,the visual detection limit of the test strips was 100 ng/m L.Combined with the gold standard reading instrument,the linear detection range was 1-1000 ng/m L,and the LOD value was 0.027 ng/m L.The method showed specific recognition ability for MG.RSDs of the test strips ranged from 8.77%to 10.18%in batches and from 7.92%to 13.59%in batches.The recoveries of 100 ng/m L,1μg/m L and 5μg/m L were determined.The recoveries ranged from 94.13%to 104.22%with RSDS ranging from 7.51%to 14.28%.Conclusion:Two MG detection methods based on aptamer recognition were established in this thesis,namely,the colorimetric detection method and colloid-gold strip detection method,to explore the possibility of aptamer detection of MG in water.The two methods are based on the specific recognition of MG by aptamers,and the qualitative and quantitative analysis of MG can be completed by the colloidal gold visualization method and the combination of small detection instruments.The former is mainly used to verify the recognition function of aptamers and MG.Both methods are novel MG detection methods with low cost,simple operation,visual detection,high efficiency and convenience,and easy popularization.The aptamer-based colloidal gold strip is worthy of further research and optimization and has great potential and development prospects for commercial products. |
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