Study On Fluorescence Detection Of Ochratoxin A Based On Strand Displacement Amplification Technology

ObjectiveOchratoxin A(OTA)is a kind of mycotoxin with wide distribution,strong toxicity,high stability and great harm.The pollution of OTA in food has become a public health food safety problem.Continuous innovation and enrichment of OTA detection methods is an important measure to effectively ensure human food safety,which has important practical significance.In this study,magnetic nanoparticles(MNPs)as solid phase carriers,aptamers(Apt)as recognition molecules,and then combined with strand displacement amplification(SDA)to establish two new methods for the detection of OTA,which provided a new choice for the detection of OTA in food and a new idea for the establishment of other trace small molecule detection methods.Methods1.Fluorescence detection of OTA based on linear aptamer and strand displacement amplification technologyFirstly,the biotinylated OTA aptamers reacted with streptavidin magnetic beads to obtain MNPS-Apt.Secondly,the complementary DNA strand-1(c DNA-1)was added,and after double-strand hybridization,c DNA-1 was bound to MNPs-Apt,magnetically separated,and the supernatant was discarded.When OTA was present,OTA preferentially binded to Apt to compete for c DNA-1 and re-disintegrated it in solution.At this time,the supernatant was collected by magnetic separation,and the amount of c DNA-1 in the supernatant was positively correlated with OTA.Then,added molecular beacon-1,Klenow fragment,DNA primers,and deoxy ribonucleoside triphosphate(d NTP)for SDA reaction to amplify the fluorescence signal.Finally,the fluorescence signal was detected with a fluorescence spectrophotometer.After optimizing some experimental conditions,the fluorescence quantitative method of OTA was established.2.Fluorescence detection of OTA based on hairpin aptamer and strand displacement amplification technologyOn the basis of the first method,a hairpin aptamer(Hap)was constructed by connecting Apt,extended strand and c DNA-2 in turn,which simplified the experimental steps.In addition,a multifunctional microplate reader was used instead of fluorescence spectrophotometer to realize the high-throughput and rapid detection of OTA.When OTA was present,Apt in Hap captured it and released c DNA-2,which was used as a hybridization site for molecular beacon-2,and then SDA reaction was carried out to achieve signal amplification.On the contrary,the hairpin structure could not be opened,the molecular beacon-2 did not hybridize with c DNA-2,the SDA reaction could not be carried out,and the final fluorescence signal was weak.After optimizing some experimental conditions,the fluorescence quantitative method of OTA was established.3.Methodological comparison and sample analysisThe gold standard high performance liquid chromatography(HPLC)and the above two new methods were used to detect the spiked samples of wheat flour and corn flour,and the detection limit,linear range,precision and accuracy of the three methods were compared.Results1.Fluorescence detection of OTA based on linear aptamer and strand displacement amplification technology(1)Coupling and characterization of MNPs and Apt:The optimal concentrations of MNPs and APT were 1.3 mg/m L and 300 nmol/L,respectively.Magnetic properties,transmission electron microscopy and Zeta potential characterization showed that MNPs-Apt had strong magnetic properties,and the suspension was stable.(2)The characterization of capturing OTA by MNPs-Apt:The content changes of OTA in the solution before and after adding MNPs-Apt showed that MNPs-Apt could capture OTA effectively,and the optimal dosage of MNPs-Apt stock solution was 65?L.(3)Feasibility analysis:In a certain concentration range,the fluorescence intensity was obviously increased by SDA reaction,and the limit of detection was reduced.(4)Conditions optimization:The sequence of c DNA-1(A),0.08 U/m L Klenow fragment,250 nmol/L primer,120 min SDA reaction time were selected as the optimal experimental conditions.(5)Standard curve and methodology evaluation:In the range of 0.5 ng/m L?128ng/m L,?F/F₀showed a good linear relationship with the logarithm of OTA concentration.The standard curve was Y=0.535X+0.525(Y is?F/F₀,X is lg C_OTA),R²=0.994,the detection limit was 0.125 ng/m L,and the precision,accuracy and specificity were all good.2.Fluorescence detection of OTA based on hairpin aptamer and strand displacement amplification technology(1)Characterization of MNPs-Hap:The Zeta potential of MNPs-HAap was-19.5 m V,indicating that the suspension was stable.(2)Feasibility analysis:The results of pre experiment showed that the difference of the results of 0,1,10 and 100 ng/ml OTA standard solution was obvious,and the method was feasible.(3)Conditions optimization:The sequence of Hap2,25?L MNPs-Hap,buffer solution with p H 7.4,0.06 U/m L Klenow fragment were selected as the optimal experimental conditions.(4)Standard curve and methodology evaluation:In the range of 2 ng/m L?64ng/m L,?RFU/RFU₀showed a good linear relationship with the logarithm of OTA concentration.The standard curve was Y=0.740X+0.378(Y is?RFU/RFU₀,X is lg C_OTA),R²=0.991.The detection limit was 0.5 ng/m L,and the precision,accuracy and specificity were all good.3.Methodological comparison and sample analysisThe limits of detection of linear aptamer and hairpin aptamer fluorescence were0.125 ng/m L and 0.5 ng/m L,respectively,which were lower than that of HPLC(5ng/m L).The relative standard deviation(RSD)of sample by HPLC was1.48%?9.71%,and the recovery was 94.33%?98.99%.The RSD of linear aptamer fluorescence method was 6.23%?17.57%,the recovery was 85.45%?108.35%.The intra-assay RSD of the hairpin aptamer fluorescence method was 4.62%?11.88%,the recovery was 90.60%?105.27%,and the inter-assay RSD was 6.17%?11.49%,the recovery was 90.48%?106.72%.ConclusionThese two new methods of detection OTA established by the study have the advantages of high sensitivity,good accuracy,strong specificity,economical environmental protection,simple and fast.They are expected to be used in the detection of mycotoxins in food,and have potential practical application value in ensuring food safety.