Screening Aptamer For Agaricus Bisporus Lectin And Establishment Of Detection Method

Agaricus bisporus lectin(ABL)is one of the anti-nutritional factors in A.bisporus.As a major potential allergen,it can cause allergic reactions in the skin,stomach,and intestines;in severe cases,it can cause asthma attacks,anaphylactic shock,life-threatening and even death.At present,due to the allergen labeling regulations of packaged foods are not yet complete,food allergen detection technology is essential to prevent foods containing allergens from entering the circulation field and reduce the occurrence of allergic incidents.Currently,the testing methods for allergens include:immunological detection technology,polymerase chain reaction technology,mass spectrometry technology,and bioresonance technology,the above methods have their own advantages and disadvantages.Due to many shortcomings of existing detection methods,it is extremely urgent to establish a simple and sensitive detection method for ABL in food.Nucleic acid aptamers are known as "chemical antibodies".It is a single-stranded oligonucleotide sequence with high affinity and strong specific binding to the target molecule,which is obtained by the systematic evolution of ligands by exponential enrichment(SELEX).It has a stable secondary structure and a special three-dimensional conformation.In this study,the subtractive-SELEX method was used to screen the aptamers of ABL.The aptamers were used as specific recognition probes and gold nanoparticles were used as indicators to establish a new detection method for the detection of ABL by aptamer nanogold colorimetry.The subtractive-SELEX method was used to screen ABL aptamers.This article was based on the fact that A.bisporus lectin is a glycoprotein,and its hydrophobic effect can be used to adsorb to the microtiter plate to fix it on the solid matrix.Thus,a strategy for screening aptamers of A.bisporus lectin was developed.The oligonucleotide library with 20 nt primer sequence each end and 40 nt random bases sequence in the middle was synthesized.After incubating the pretreated random library with the subtractive target(bovine serum albumin,BSA),then incubated with the target(ABL)to form an ABL-ssDNA complex,which was retained on the well.The target-bound ssDNA was collected by heating denaturation.Next,the single strands were recovered by ethanol precipitation and used as templates for PCR amplification.The secondary library was prepared by the streptavidin magnetic bead method,and 13 rounds of screening were performed to obtain the aptamer group.The products of the final round screening were cloned and sequenced to obtain 43 sequences,which contained multiple high-frequency sequences.After bioinformatics analysis of the sequences,6 candidate aptamers were selected.The affinity and specificity were identified by the enzyme-linked aptamer method.The results showed that seq-41 was the optimal aptamer.Based on aptamers and gold nanoparticles with color-changing effect,a visual detection method of ABL was developed.Nano-gold were synthesized by sodium citrate reduction method.The monodispersed gold nanoparticles were wine-red in the solution.The aptamer and the gold nanoparticles combined with each other under the action of electrostatic attraction,and aptamers protected the gold nanoparticles against salt induction and prevented the particles from agglomeration and discoloration.Since the ABL aptamers bound specifically to ABL,when ABL was added to the solution,ABL competed for binding to aptamers bound to nano-gold.The exposed gold nanoparticles agglomerated under the action of high salt concentration,and the color of the solution changed from red to blue-violet.Under the optimal experimental conditions,the concentration of ABL had a good linear relationship with A620/A520.The regression equation was y=0.4146x+0.4307(R2=0.9964),the linear range was 0.08?1.70 ?g/mL,and the detection limit was 0.062 ?g/mL.The specificity experiment proved that this method had strong specificity for ABL.The recovery test of adding standard sample verified that the recovery rate of this method in food matrix was 92.4%?105.3%.The above research results showed that this method was simple in design,easy to operate,highly sensitive and specific,and could provide a new reference for the detection of allergens in edible fungi and plants.At the same time,it provided technical support for allergy prevention and the development of desensitized food.