Study On The Rapid Detection Of Mycotoxins By Aptamer-based Lateral Flow Chromatography Test Strips

Mycotoxins have great harm to human health.It has been a hot topic in the field of food safety to seek a rapid and highly sensitive mycotoxin detection technology.Aptamers are DNA or RNA single strands produced from random nucleic acid libraries by exponential enrichment ligands system evolution technology.They can be folded into high-level structures with high specificity and affinity to identify specific targets.In this study,a method for the detection of type-B aflatoxins and a rapid method for the simultaneous determination of ochratoxin A（OTA）and aflatoxin B₁（AFB₁）were established by using test strip and the competitive mode of aptamer complementary chain;the solid phase extraction（SPE）column was used to purify the samples in one step,which provided theoretical basis and technical support for the rapid detection of mycotoxins.The main research contents are as follows:1.A SPE column was developed for the extraction and purification of mycotoxins.One-step purification of OTA and type-B aflatoxins was realized by optimizing extraction reagents and purification conditions through the SPE column.The recovery rate of OTA and type-B aflatoxins was 92.4%-102.9%by using the SPE column to purify the sample,and the relative standard deviation（RSD）was 1.0%-4.3%.It was greatly improved the efficiency of sample pretreatment by using the SPE column,which was beneficial for the rapid detection of mycotoxins.2.A method for the detection of type-B aflatoxins by fluorescence side flow chromatography was developed.This method was based on the competition between aflatoxins of type-B aflatoxins and complementary DNA of the Cy5 labeled aptamer.The sensitivity of detecting type-B aflatoxins was improved by optimizing the length and concentration of aptamers,complementary probes at the test line（T line）,the type and ion concentration of buffer,and the proportion of organic solvents.The complementary chain of aptamers was first used as the T line to construct the aptamer test strip for the rapid,quantitative and specific detection of type-B aflatoxins,and the linear range of this method was 0.2-20 ng/m L with the detection limit of 0.16 ng/m L.The recovery was 93.3%-112.0%and the RSD was 2.7%-7.9%in peanut,almond and dried fig samples.which was consistent with the accuracy of the HPLC-MS/MS method.This method has the advantages of low cost,fast analysis speed and convenient operation.It provides a new rapid detection technology for rapid detection of type-B aflatoxins in the field,and has a good application prospect.3.An aptamer multi-residue test strip method for simultaneous detection of OTA and AFB₁ was established.The method was based on the competition of the aptamers between OTA and AFB₁ and the complementary chain of the aptamer.The sensitivity and accuracy of simultaneous detection of OTA and AFB₁ were improved by optimizing the concentration of aptamer and p H value of buffer system.The method had a good linearity of 0.5-50 ng/m L with OTA and AFB₁,and the correlation coefficient R₂ was 0.9887 and 0.9910,and the detection limit was 0.51 ng/m L and 0.38 ng/m L,respectively.The recoveries of OTA and AFB₁ in peanut and raisin were 82.1%-109.7%and 83.3%-110.1%,respectively,and the RSD was 1.9%-8.2%,which was proved by the HPLC-MS/MS.The method provided theoretical basis and technical support for the simple and sensitive detection of OTA and AFB₁.