Development Of A Microsphere Array Technology For Multiply Dctcction Of Toxic Fungi And Its Toxins

The toxins produced by fungi,the eukaryotic microorganisms,are called mycotoxins thatbelong to poisonous secondary metabolites. The contamination of toxin producing fungi andtheir mycotoxins on agricultural products, food stuffs as well as animal feeds becomeincreasingly severe and lead to healthy harms and economic losses. Among them, theproblems caused by aflatoxin B1(AFB1) and zearalenone (ZEN) and these toxin producingfungi are more obvious. Therefore, developing newly fast detection methods for abovetoxins and fungi is very necessary. Multi-Analyte Suspension Array(MASA) was firstdeveloped by American Luminex Company in1990's. This new technology has theadvantages of high throughout, flexible, highly sensitive, well repeatable and rapidcharacteristics for detection. The present paper aims to develop new detection methods for thetwo toxins and the toxin-producing fungi with the microsphere based suspension arraytechnology platform, the related reports has yet to be seen, the studies conducted as it follows:1. Succeeding in synthesizing artificial antigen: AFB1-OVA and ZEN-OVA by usingmycotoxin hapten and preparing the AFB1and ZEN polyclonal antibodies that meet therequirement of the multiply quantitative microsphere array technology.The AFB1-OVA and the ZEN-OVA have been used for immunizing New Zealand whiterabbits and collecting compliance titer antiserum. After using the improved the bitterness-saturated ammonium sulfate precipitation purification, the SDS polyacrylamide gelelectrophoresis for identifying the purity of the antibody showed that electrophoretic bandswas reduced but became more clear and obvious. After testing, the titers of the purified AFB1and ZEN polyclonal antibodies were1:32000and1:700; the protein concentrations were6.97mg/mL and6.42mg/mL; the affinity constants of6.89×108L/mol and1.64×107L/mol,respectively. The results of the cross-reactivity test indicated that the cross-reactivity ratebetween AFB1polyclonal antibody and aflatoxin B2was16.05%, the cross-reactivity ratebetween AFB1polyclonal antibody and zearalenone(ZEN) was6.66%. No cross reactivityhas been found between the ZEN polyclonal antibody and the AFB1.2. For the first time in the world, the technology was introduced into the detection field for toxin producing fungi and the hapten toxins produced. Using the AFB1polyclonal andmonoclonal antibodies, the ZEN polyclonal and monoclonal antibody, more than10,000experimental samples were tested and analyzed, and a model established was shown that itcould be used to optimize the reaction of AFB1and ZEN in the microsphere arraytechnology for the single quantitative detection and the multiple quantitative detection ofAFB1and ZEN.1) The best reaction system for the AFB1detection with the technology was described below:the coupled antigen of AFB1-BSA was100μg, the AFB1and antibody incubation time was24h, the signal and multiple critical saturated concentration of AFB1were0.75μg/mL and6.0μg/mL respectively. The best reaction system for the ZEN was described below: thecoupled antigen of AFB1-BSA was100μg, the AFB1and antibody incubation time was15h,the signal and multiple critical saturated concentrations of AFB1were2.0μg/mL and10.0μg/mL, respectively.2) A. Using the AFB1monoclonal antibody, the AFB1: R2value of the standard curvecreated by the14thcoding microsphere was0.9734, the IC50,2.33ng/mL, the minimumdetection value,0.1165ng, with the linear range of0.06-11.25ng/mL. Using the AFB1monoclonal antibody, the AFB1: R2value of the standard curve created by the28thcodingmicrosphere was0.9734, the IC50,3.00ng/mL, the minimum detection value,0.1500ng, withthe linear range of0.03-11.25ng/mL. The results of the two microspheres were notstatistically significant. B. Using the AFB1polyclonal antibody, the AFB1: R2value of thestandard curve created by the14thcoding microsphere was0.9842, the IC50,1.33ng/mL, theminimum detection value,0.0665ng, with the linear range of0.03-11.25ng/mL. Using theAFB1monoclonal antibody, the AFB1:R2value of the standard curve created by the28thcoding microsphere was0.9906, the IC50,1.43ng/mL, the minimum detection value,0.0715ng, with the linear range of0.03-11.25ng/mL. The results of the two microsphereswere not statistically significant.3) A. Using the ZEN monoclonal antibody, the ZEN: R2value of the standard curve createdby the36thcoding microsphere was0.9936, the IC50,,1.3846ng/mL, the minimum detectionvalue,0.0692ng, with the linear range of0.05-10.0ng/mL. B. Using the ZEN polyclonal antibody, the ZEN: R2value of the standard curve created by the36thcoding microsphere was0.9988, the IC50,3.2500ng/mL, the minimum detection value was0.1625ng, with the linearrange of0.05-10. ng/mL.4) Using the AFB1polyclonal antibody and the ZEN monoclonal antibody, the28thand the36thcoding microspheres were tested by the multiple quantitative detection. As a result, theAFB1: R2value of the standard curve created was0.9842, the IC50,2.3333ng/mL, theminimum detection value,0.05833ng, with the linear range of0.06-4.8ng/mL. The ZEN: R2value of the standard curve created was0.997, the IC50,3.2000ng/mL, the minimum detectionvalue,0.0800ng, with the linear range of0.10-10.0ng/mL.5) Using the AFB1polyclonal antibody response system, the recovery rates of the imitatingcontaminated skim milk and full milk were all greater than75%, with the coefficient ofvariation less than5%.6) Using the ZEN monoclonal antibody response system, the recovery rates of the imitatingcontaminated skim milk and full milk were all greater than75%, with the coefficient ofvariation less than5%.7) Using the multiply quantitative detection of AFB1and ZEN established on the microspherearray technology platform to test the APFB1and ZEN simultaneously, the recovery rates ofthe imitating contaminated skim milk and full milk were all greater than75%, with thecoefficient of variation less than5%.3. This study established the multiple detection system for the hidden aflatoxin andzearalenone producing fungi, which was5to500times sensitive than the traditionaldetection method, filling the blank of simultaneous detection of these fungi.1) The NCBI identified the conservative DNA region in the aflatoxin producing control genenor-1, ver-1, omt-A and ZEN producing control gene PKS4, ZEB2as well as the genes ITS.Then, the conservative DNA regions were then tested using the Premier5.0software, anonline alignment tool for multiple sequence alignment. The results generated six primers andprobes that have valid sequence and specificity and low cross reactivity rate.2) The six target fragments, nor-1, ver-1in omtA, PKS4, ZEB2, and ITS, were recollected,clone constructed, positively clones sequenced. The results of sequencing showed that: the similarity of the nor-1in target fragments and in Genbank Aspergillus flavus and A.parasiticus was100%and98%, respectively. The homology of the ver-1in target fragmentsand in A. flavus and A. parasiticus,100%and97%, respectively. The similarity of the nor-1intarget fragments and in A. flavus and A. parasiticus.100%and94%, respectively. Thesimilarity of the PKS4and ZEB2in target fragments and in Genbank Fusarium graminearumwas100%. The homology of the ITS in target fragments and in A. flavus, A.fumigatus and A.niger were100%. The results above demonstrated the good specificity the primers and theprobes.3) Based on the microsphere array technology, the downstream primer was biotin-modified tomake the PCR amplification products biotinylated. The probes were aminated to coupleprobes with carboxylated coding microspheres. Coupling quality control sequences matchingthe nucleic probes were synthesized and their5'end was biotin-labeled for the results ofeffectively coupling between nucleic acid probe and fluorescent coded microspheres.4) By using the self-designed primers and probes, this study established multiple PCRreaction system, and the multiple detection of the aflatoxin and zearalenone producing hiddenfungi. The positive results were generated in A, flavus, A, parasiticus and F,graminearum.The negative results were found in A. nidulans, A. terreus, A. fumigatus and A. niger. Theresults above demonstrated the good specificity the primers and the probes. The method couldsimultaneously detect single, double, triple, tetra-, quinta-,hexa-targets of the permutationand combination of the nor-1, ver-1, omt-A, PKS4, ZEB2and ITS. Sensitivity tests usingpositive clones plasmid showed that the detection sensitivity of nor-1, ver-1, omt-A, PKS4,ZEB2and ITS was0.20ng/PCR,2.00pg/PCR,0.20ng/PCR,0.02ng/PCR,2.00pg/PCR,2.00pg/PCR respectively.