【作者】 宋慧君；

【导师】 曹远银；

【作者基本信息】 沈阳农业大学 ， 有害生物与环境安全， 2012， 博士

【摘要】 能够产生真菌毒素的真核细胞型微生物即为产毒真菌，真菌毒素是其产生的具有毒性的二级代谢产物。产毒真菌及其产生的毒素对农作物产品、食品及饲料的污染日趋严重，造成了危害与损失，其中，黄曲霉毒素和玉米赤霉烯酮及其产毒真菌问题比较突出，因此研发上述两类毒素和产毒真菌的快速检测新技术十分必要。液相芯片是美国Luminex公司开发出的将流式检测技术与芯片技术有机结合了的一种新技术，该技术具有高通量性、灵活性大、敏感性高、重复性好、检测速度快等优点。本文针对目前在产毒真菌和真菌毒素检测领域里未见液相芯片技术的研究报道的问题，开展了如下研究。一、成功使用小分子真菌毒素OVA偶联物为免疫抗原，制备出了可以满足液相芯片检测实验的AFB1和ZEN多克隆抗体，具有一定的先进性。分别以人工合成抗原AFB1-OVA和ZEN-OVA为免疫原免疫新西兰大白兔，收集效价达标的抗血清；用改进的辛酸-饱和硫酸铵沉淀法纯化，SDS聚丙烯凝胶电泳纯度鉴定，其抗体显示出泳带数减少但泳带更明显清晰。经测定，纯化的AFB1和ZEN多克隆抗体效价分别为1：32000和1：700；蛋白浓度分别为6.97mg/mL和6.42mg/mL；亲和常数分别为6.89×108L/mol和1.64×107L/mol；交叉反应试验证明：AFB1多克隆抗体分别与黄曲霉毒素B2、G1的交叉反应率为16.05%和6.66%；ZEN多克隆抗体与AFB1无交叉反应。二、首次将液相芯片技术引入真菌毒素检测领域，利用AFB1多克隆抗体和单克隆抗体、ZEN多克隆抗体和单克隆抗体，通过对近万个实验样本的分析测试，优化得到了AFB1、ZEN液相芯片反应体系，建立了AFB1、ZEN免疫液相芯片单重、二重测试方法，填补了液相芯片在真菌毒素研究领域的空白。1．研究确定了AFB1液相芯片最佳反应体系为：偶联抗原AFB1-BSA为100μg，AFB1与抗体孵育时间为24h，AFB1单抗和多抗的临界饱和浓度分别为0.75μg/mL和6.0μg/mL；ZEN液相芯片最佳反应体系为：偶联抗原ZEN-BSA为100μg，ZEN与抗体孵育时间为15h，ZEN单抗和多抗的临界饱和浓度分别为2.0μg/mL和10.0μg/mL。2．1采用AFB1单克隆抗体，14号微球所建立的AFB1检测标准曲线R2为0.9734，IC50为2.33ng/mL，最低检测量为0.1165ng，线性范围为0.06-11.25ng/mL；采用AFB1单克隆抗体，28号微球所建立的AFB1检测标准曲线R2为0.9762，IC50为3.00ng/mL，最低检测量为0.1500ng，线性范围为0.03-11.25ng/mL；两种微球之间无显著性差异。2．2采用AFB1多克隆抗体，14号微球所建立的AFB1检测标准曲线R2为0.9842，IC50为1.33ng/mL，最低检测量为0.0665ng，线性范围为0.03-11.25ng/mL；采用AFB1多克隆抗体，28号微球所建立的AFB1检测标准曲线R2为0.9906，IC50为1.43ng/mL，最低检测量为0.0715ng，线性范围为0.03-11.25ng/mL；两种微球之间无显著性差异。3．1采用ZEN单克隆抗体，36号微球所建立的ZEN检测标准曲线R2为0.9936，IC50为1.3846ng/mL，最低检测量为0.0692ng，线性范围为0.05-10.0ng/mL；3．2采用ZEN多克隆抗体，36号微球所建立的ZEN检测标准曲线R2为0.9988，IC50为3.2500ng/mL，最低检测量为0.1625ng，线性范围为0.05-10.0ng/mL。4．采用AFB1多克隆抗体、ZEN单克隆抗体，28号和36号微球建立AFB1、ZEN二重定量检测方法，其中检测AFB1标准曲线R2为0.9842，IC50为2.3333ng/mL，最低检测量为0.0583ng，线性范围为0.06-4.80ng/mL；检测ZEN标准曲线R2为0.997，IC50为3.2000ng/mL，最低检测量为0.0800ng，线性范围为0.10-10.0ng/mL。5．以脱脂牛奶和全脂牛奶为实验样本，选择AFB1多克隆抗体反应体系，进行AFB1添加回收试验，结果显示：所有样本回收率均大于75%，变异系数均小于5%。6．以脱脂牛奶和全脂牛奶为实验样本，选择ZEN单克隆抗体反应体系，进行ZEN添加回收试验，结果显示：所有样本回收率均大于75%，变异系数均小于5%。7．以脱脂牛奶和全脂牛奶为实验样本，选择AFB1、ZEN液相芯片二重定量检测反应体系，同时进行AFB1、ZEN添加回收试验，结果显示：所有样本回收率均大于75%，变异系数均小于5%。三、研制建立了潜藏性产黄曲霉毒素产毒真菌和产玉米赤霉烯酮产毒真菌液相芯片多重检测方法，其灵敏度比传统方法提高了5-500倍，并首次实现了同时检测两种产毒真菌的目标理想，填补了该领域的研究空白。1．通过NCBI进行DNA序列比对和分析，分别针对产黄曲霉毒素产毒真菌产毒控制基因nor-1、ver-1、omtA，产玉米赤霉烯酮产毒真菌产毒控制基因PKS4、ZEB2和真菌ITS基因片段的保守区域，利用Primer Premier5.0软件设计、在线序列比对工具多重序列比对，得到了序列特异性好、交叉反应率很小的6种探针引物。并以黄曲霉（AS3.4408）菌株DNA和禾谷镰刀菌（AS3.4598）菌株DNA为摸板，采用单重PCR技术，进行扩增验证。2．回收nor-1、ver-1、omtA、PKS4、ZEB2和ITS6个目的片段，进行克隆构建、阳性克隆子测序。测序结果显示该nor-1基因片段与Genbank中黄曲霉nor-1和寄生曲霉nor-1的同源性分别达100%和>98%；ver-1基因片段与黄曲霉ver-1和寄生曲霉ver-1的同源性分别达100%和97%；omtA基因片段与黄曲霉omtA和寄生曲霉omtA的同源性分别达100%和>94%。PKS4和ZEB2的基因片段分别与Genbank中禾谷镰刀菌PKS4和ZEB2的同源性均达100%。ITS片段与黄曲霉、烟曲霉、黑曲霉等序列同源性达100%。以上均证实所设计的引物特异性好。3．根据液相芯片检测方法原理，对下游引物进行生物素修饰，以使PCR扩增产物生物素化；对探针进行氨基化修饰，以便于探针与羧基化微球偶联；合成与核酸探针完全互补的偶联质控序列，并对其5’端进行生物素标记，以有效报告核酸探针与荧光编码微球偶联结果。4．采用自主设计的特异性探针与引物，建立多重PCR反应体系，建立了潜藏性产黄曲霉毒素和玉米赤霉烯酮产毒真菌液相芯片多重检测方法。结果表明：用该方法检测产黄曲霉毒素的黄曲霉、寄生曲霉，产玉米赤霉烯酮的禾谷镰刀菌的检测结果为阳性；检测不产上述两种真菌毒素的构巢曲霉、土曲霉、烟曲霉、黑曲霉的检测结果为阴性，表明所建立方法特异性好。采用阳性克隆子质粒DNA评价该方法的灵敏性，实验结果表明：该方法可对nor-1、ver-1、omtA、PKS4、ZEB2、ITS基因进行1重、2重、3重、4重、5重、6重检测，其检测灵敏度分别为：0.20ng/μL、2.00pg/μL、0.20ng/μL、0.02ng/μL、2.00pg/μL、2.00pg/μL。更多还原

【Abstract】 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.更多还原