Research On Rapid Detection Technology Of Zearalenone And T-2 Toxin Based On Isothermal Amplification

Objective:Taking Zearalenone(ZEN)and T-2 toxin as research objects,Aptamer-functionalized magnetic microspheres(AP-MMPs)were prepared to enrich the target materials.Combined with isothermal amplification technology and catalytic cleavage activity of PfAgo protein,two kinds of highly sensitive and highly specific fluorescence aptasensors were established.Methods:1.A fluorescence aptasensor for detecting zearalenone based on rolling circle amplification(RCA)and PfAgo protein was constructed.Firstly,AP-MMPs were prepared by combining aptamers with magnetic spheres.The target,AP-MMPs,and primers were added,magnetically separated,and the supernatant was collected.The loop-lock probe,T4 DNA ligase,phi 29 DNA polymerase,and d NTPs were added,to produce long single strands of RCA.At this point,the PfAgo protein is added,which is activated to cleave the target strand under the guidance of g DNAs.After cleavage,a new 5’phosphorylated secondary g DNAs is formed,and the signaling molecules containing fluorescent and quenched groups are further cleaved after the addition of signaling molecules.Finally,the signal is output by fluorescence spectrophotometer.The higher the concentration of the target,the stronger the fluorescence intensity.2.A fluorescence aptasensor technology based on hybrid chain reaction(HCR)for the simultaneous detection of ZEN and T-2 toxin was constructed.Constant temperature and enzyme free HCR was used for signal amplification,and a portable constant temperature fluorescence detector was used for signal output.First,the AP-MMPs are synthesized in the same way.When the target was present,AP-MMPs was used to enrich the target,and the free supernatant contained H0 primers.Hairpin H1(modified with fluorescent and quenched groups)and hairpin H2 are added,and H0 can open hairpin H1 and H2 to grow double chains.At this time,the distance between the fluorescent group on H1 and the quenched group is far away,and the fluorescence signal is restored.The higher the concentration of the target,the stronger the fluorescence signal.Results:1.A fluorescence aptasensor technology based on RCA and PfAgo protein for detection of ZEN:Under the optimal experimental conditions,the concentration of ZEN measured by this method showed a good linear relationship in the range of0.01～10 ng/m L.The linear equation was y=403.8691lgx+1661.3765(R~2=0.9911,3σ/k),with a detection limit of 0.0053 ng/m L.The average recovery rate of corn flour was104.58%～112.82%,and the relative standard deviation(RSD)was less than 10%.2.A fluorescence aptasensor technology based on HCR for the simultaneous detection of ZEN and T-2 toxin:Under optimal experimental conditions,T-2 and ZEN showed good fluorescence responses in the dynamic range of 0.001～10 ng/m L and0.01～100 ng/m L,with linear equations of y=777.5lgx+3099.3(R~2=0.9913,3σ/k)and y=247.5lgx+722.8(R~2=0.9916,3σ/k),respectively.The detection limits were 0.1 pg/m L and 1.2 pg/m L,respectively.The average recoveries of T-2 toxin in corn and oat were90.16%～101.40%and 87.79%～100.3%respectively;The average recoveries of ZEN in corn flour and oats were 90.7%～111.10%and 94.49%～104.89%.The RSDs of the two actual samples of the above two toxins is less than 10%.Conclusions:1.A fluorescence aptamer sensing technique based on RCA and PfAgo protein was constructed to detect ZEN.This method has a good detection range and a lower detection limit,and has realized the recovery of ZEN standard in corn meal samples.2.A fluorescent aptamer sensing technology based on HCR for simultaneous detection of T-2 and ZEN was constructed.Based on the characteristics of HCR,such as constant temperature and no enzyme,this strategy is simple in operation,and overcomes the deficiency of detecting a single target,and realizes the simultaneous detection of ZEN and T-2.