農(nóng)產(chǎn)品中四種常見(jiàn)真菌毒素免疫檢測(cè)技術(shù)研究
發(fā)布時(shí)間：2019-05-22 02:49
【摘要】：真菌毒素(Mycotoxin)是一類由真菌產(chǎn)生的小分子次級(jí)代謝產(chǎn)物,農(nóng)作物在生長(zhǎng)、收獲、貯存和運(yùn)輸?shù)拳h(huán)節(jié)均易受到真菌毒素污染,現(xiàn)已發(fā)現(xiàn)400多種真菌毒素,大部分具有致癌、致畸和致突變作用且混合污染時(shí)毒性會(huì)顯著增強(qiáng)。真菌毒素可通過(guò)污染谷物和飼料或經(jīng)動(dòng)物源性食品進(jìn)入食物鏈,對(duì)人類和動(dòng)物健康造成嚴(yán)重傷害,近年來(lái)已成為食品安全領(lǐng)域的研究重點(diǎn)。真菌毒素現(xiàn)有檢測(cè)方法包括生物鑒定法、化學(xué)分析法、儀器分析法和免疫分析法等。生物鑒定法專一性不強(qiáng)；化學(xué)分析法精確度低；儀器分析靈敏度高,可準(zhǔn)確定量,但樣本前處理復(fù)雜并且檢測(cè)儀器昂貴,難以在基層實(shí)驗(yàn)室推廣使用。免疫分析方法快速、靈敏度高、特異性好,并且成本較低,近年來(lái)在真菌毒素檢測(cè)領(lǐng)域發(fā)展迅速。本課題選取農(nóng)產(chǎn)品中常見(jiàn)且危害較大的黃曲霉毒素B1 (Alfatoxin B1, AFB1)、赭曲霉毒素A (Ochratoxin A, OTA)、伏馬毒素B1 (Fumonisin B1, FB1)和玉米赤霉烯酮(Zeralenone, ZEN)為檢測(cè)對(duì)象,以單克隆抗體作為生物識(shí)別元件,基于競(jìng)爭(zhēng)ELISA和免疫層析原理,結(jié)合信號(hào)放大、免疫傳感、抗體芯片等技術(shù),建立多種真菌毒素的快速、敏感、高通量免疫檢測(cè)方法。本論文的主要研究?jī)?nèi)容和結(jié)果如下：1.赭曲霉毒素A的間接競(jìng)爭(zhēng)ELISA定量檢測(cè)技術(shù)對(duì)OTA單克隆抗體2D8的免疫學(xué)性質(zhì)進(jìn)行鑒定,以此抗體為基礎(chǔ)建立間接競(jìng)爭(zhēng)酶聯(lián)免疫檢測(cè)法(ci-ELISA)�？贵w2D8效價(jià)為1：1.024×106,親和常數(shù)Ka=3.75×109L/M,重鏈為IgG1型,與赭曲霉毒素B (Ochratoxin B, OTB)的交叉反應(yīng)率為5.4%,與AFB1、FB1、ZEN、桔青霉毒素(Citrinin, CIT)、展青霉毒素(Patulin, PAT)和嘔吐毒素(Deoxynivalenol, DON)均無(wú)交叉反應(yīng)。建立的ci-ELISA檢測(cè)限為0.08 ng/mL,檢測(cè)范圍0.12-1.18 ng/mL,加標(biāo)回收率可達(dá)91.2-110.3%,與商品化ELISA試劑盒相關(guān)性較好,可用于谷物及飼料樣本中OTA的定量檢測(cè)與分析,也為后續(xù)其它免疫檢測(cè)方法的建立奠定基礎(chǔ)。2.基于免疫磁珠和生物素-鏈霉親和素的間接競(jìng)爭(zhēng)ELISA定量檢測(cè)玉米赤霉烯酮為縮短反應(yīng)時(shí)間,提高檢測(cè)靈敏度,以納米磁珠作為固相載體包被ZEN單克隆抗體,基于競(jìng)爭(zhēng)反應(yīng)原理,利用生物素-鏈霉親和素系統(tǒng),建立新型酶聯(lián)免疫檢測(cè)方法(MNP-bsELISA).該方法檢測(cè)限為0.04 ng/mL,檢測(cè)范圍0.07-2.41ng/mL,加標(biāo)回收率為92.8-111.9%,變異系數(shù)小于10%,對(duì)天然樣本中ZEN的定量結(jié)果與LC-MS/MS方法具有良好的相關(guān)性。3.赭曲霉毒素A磁珠-電化學(xué)免疫傳感檢測(cè)技術(shù)優(yōu)化以磁珠為固相載體的免疫競(jìng)爭(zhēng)反應(yīng),選取最佳模式并結(jié)合電化學(xué)信號(hào)檢測(cè)技術(shù),建立了OTA電化學(xué)免疫傳感檢測(cè)技術(shù)。該方法的檢測(cè)下限達(dá)到0.007ng/mL,檢測(cè)范圍為0.01-0.82 ng/mL,加標(biāo)回收率為78.7-91.6%,標(biāo)準(zhǔn)偏差和變異系數(shù)均小于15%,穩(wěn)定性較好,可實(shí)現(xiàn)樣本中OTA的痕量檢測(cè)。4.赭曲霉毒素A和玉米赤霉烯酮二聯(lián)免疫層析試紙條研制基于免疫層析原理,分別建立了二聯(lián)定性和定量膠體金免疫層析檢測(cè)方法用于谷物和飼料中OTA和ZEN的檢測(cè)。其中二聯(lián)定性膠體金免疫層析試紙條對(duì)OTA和ZEN的檢測(cè)限分別為0.625 ng/mL和1.25 ng/mL,二聯(lián)定量膠體金免疫層析試紙條對(duì)OTA的檢測(cè)限為0.25 ng/mL,檢測(cè)區(qū)間為0.32-12.16 ng/mL;對(duì)ZEN的檢測(cè)限為0.5 ng/mL,檢測(cè)區(qū)間為0.58-39.72 ng/mL;兩種真菌毒素在玉米、小麥和飼料樣品中加標(biāo)回收率為79.6-105.2%。制備的二聯(lián)免疫層析試紙條對(duì)樣本的檢測(cè)結(jié)果與LC-MS/MS相關(guān)性較好,可分別滿足樣本中OTA和ZEN的快速定性檢測(cè)與定量分析。5.同時(shí)檢測(cè)四種真菌毒素的高通量抗體芯片定量技術(shù)真菌毒素混合污染情況較為常見(jiàn)且毒性會(huì)顯著增強(qiáng),為對(duì)樣本中四種常見(jiàn)真菌毒素(AFB1、OTA、FB1和ZEN)進(jìn)行高通量、同時(shí)定量檢測(cè),以硝酸纖維素膜為基板,利用生物素.鏈霉親和素系統(tǒng),建立抗體芯片檢測(cè)模式。該方法對(duì)AFB1、OTA、FB1、ZEN的檢測(cè)限分別為0.21、0.19、0.24和0.09 ng/mL,檢測(cè)區(qū)間分別為0.47-55.69、0.48-127.11、0.56-92.57和0.22-31.36 ng/mL。在實(shí)際樣本中的回收率為79.2-113.4%,變異系數(shù)小于10%,穩(wěn)定性較好,可對(duì)真菌毒素進(jìn)行多重定量檢測(cè),在真菌毒素混合污染監(jiān)測(cè)領(lǐng)域具有廣闊的應(yīng)用前景。綜上所述,本研究將真菌毒素特異性單克隆抗體與磁性納米材料、生物素鏈霉親和素、電化學(xué)、芯片等信號(hào)放大和高通量檢測(cè)體系相結(jié)合,分別建立了ci-ELISA、MNP-bsELISA、磁珠電化學(xué)免疫傳感器、二聯(lián)定性和定量膠體金免疫層析試紙條和抗體芯片多重檢測(cè)技術(shù),可滿足農(nóng)產(chǎn)品中真菌毒素快速便捷、高靈敏和高通量的檢測(cè)需求,為農(nóng)產(chǎn)品、食品和飼料中真菌毒素污染監(jiān)測(cè)體系的建立提供技術(shù)支撐,也為其他小分子分析物的檢測(cè)提供參考與策略。
[Abstract]:Mycotoxin is a kind of small-molecule secondary metabolite produced by the fungus, and the crops are easy to be polluted by the mycotoxin in the aspects of growth, harvesting, storage and transportation, and more than 400 mycotoxins have been found, most of which are carcinogenic, The toxicity of teratogenic and mutagenic effects and mixed contamination is significantly enhanced. The mycotoxin can enter the food chain through the pollution of grain and feed or animal-derived food, and has serious harm to human and animal health, and has become the research focus in the field of food safety in recent years. The existing detection methods of mycotoxins include the biological identification method, the chemical analysis method, the instrument analysis method and the immunoassay method, and the like. The biological identification method is not strong in specificity, the chemical analysis method is low in precision, the analytical sensitivity of the instrument is high, the accuracy can be accurately quantified, the pretreatment of the sample is complicated and the detection instrument is expensive, and the use is difficult to be popularized in the basic-layer laboratory. The immune analysis method is rapid, high in sensitivity, good in specificity and low in cost, and has developed rapidly in the field of mycotoxin detection in recent years. Aflatoxin B1 (AFB1), aspergillotoxin A (OTA), fumonisin B1 (FB1) and Zealenone (ZEN), which are common and harmful to the agricultural products, are selected as the detection target, and the monoclonal antibody is used as the biological identification element. Based on the principle of competitive ELISA and immunochromatography, the rapid, sensitive and high-throughput immunodetection method of various mycotoxins is established by combining the techniques of signal amplification, immunosensing, antibody chip and the like. The main contents and results of this thesis are as follows:1. The immunological properties of the OTA monoclonal antibody 2D8 were identified by indirect competitive ELISA. The indirect competitive enzyme-linked immunosorbent assay (CI-ELISA) was established based on the antibody. The antibody 2D8 has a potency of 1: 1.024-106, a affinity constant Ka = 3.75-109L/ M, a heavy chain of an IgG1 type, a cross-reaction rate of 5.4% with an aspergiloxin B (Ochratoxin B, OTB), and AFB1, FB1, ZEN, Penicillium citrinin (CIT), Paecilomyces (Patulin, PAT), and a vomiting toxin (Deoxynivalenol, DON) No cross-reaction. The detection limit of ci-ELISA is 0.08 ng/ mL, the detection range is 0.12-1.18 ng/ mL, the recovery rate of the spike can reach 91.2-110.3%, the correlation with the commercial ELISA kit is good, the quantitative detection and analysis of the OTA in the grain and the feed sample can be used, and the foundation for the establishment of the subsequent other immunodetection methods is also provided. the method is based on the indirect competitive ELISA of the immunomagnetic beads and the biotin-streptavidin to quantitatively detect the corn trichoderma ketene to shorten the reaction time, improve the detection sensitivity, coat the ZEN monoclonal antibody as a solid phase carrier by using the nano magnetic beads as a solid phase carrier, and based on the principle of the competition reaction, A new enzyme-linked immunosorbent assay (MNP-bsELISA) was established using a biotin-streptavidin system. The detection limit of the method is 0.04 ng/ mL, the detection range is 0.07-2.41 ng/ mL, the recovery of the spike is 92.8-111.9%, the coefficient of variation is less than 10%, and the quantitative result of ZEN in the natural sample has a good correlation with the LC-MS/ MS method. The immune competitive reaction of the magnetic bead as the solid-phase carrier is optimized by using the magnetic bead-electrochemical immunosensing detection technology of the aspergillosis toxin A. The best mode is selected and the electrochemical signal detection technology is combined to establish the OTA electrochemical immunosensing detection technology. The detection limit of the method is 0.007 ng/ mL, the detection range is 0.01-0.82 ng/ mL, the recovery rate of the spike is 78.7-91.6%, the standard deviation and the coefficient of variation are all less than 15%, the stability is good, and the trace detection of the OTA in the sample can be realized. Based on the principle of immunochromatography, an immunochromatographic method for the detection of OTA and ZEN in grain and feed was established based on the principle of immunochromatography. The detection limit for OTA and ZEN is 0.625 ng/ mL and 1.25 ng/ mL respectively. The detection limit of the two-linked qualitative colloidal gold immunochromatographic test strip to OTA is 0.25 ng/ mL, the detection interval is 0.32-12.16 ng/ mL, the detection limit to ZEN is 0.5 ng/ mL, and the detection interval is 0.58-39.72 ng/ mL; The recovery of two mycotoxins in corn, wheat and feed samples was 79.6-105.2%. The detection results of the prepared two-linked immunochromatographic test strip and the LC-MS/ MS are better than that of the LC-MS/ MS, and the rapid qualitative detection and quantitative analysis of OTA and ZEN in the samples can be respectively met. the high-flux antibody chip quantitative technical mycotoxin mixed pollution condition of the four mycotoxins is detected at the same time, and the toxicity is obviously enhanced, and the high-flux antibody chip quantitative detection is carried out on the four common mycotoxins (AFB1, OTA, FB1 and ZEN) in the sample, Taking the nitrocellulose membrane as a substrate, and using biotin. And the streptavidin system is used for establishing an antibody chip detection mode. The detection limits of AFB1, OTA, FB1 and ZEN were 0.21, 0.19, 0.24 and 0.09 ng/ mL, respectively. The detection interval was 0.47-55.69, 0.48-127.11, 0.56-92.57 and 0.22-31.36 ng/ mL, respectively. The recovery rate of the actual sample is 79.2-113.4%, the coefficient of variation is less than 10%, the stability is good, and the mycotoxin can be tested for multiple quantitative detection, and has wide application prospect in the field of the mixed pollution monitoring of the mycotoxin. in conclusion, that present study combine the specific monoclonal antibody of the mycotoxin with the signal amplification and high-flux detection system of the magnetic nano-material, the biotin-streptavidin, the electrochemistry, the chip and the like, and respectively establish the ci-ELISA, the MNP-bsELISA and the magnetic bead electrochemical immunosensor, the two-way qualitative and quantitative colloidal gold immunochromatographic test strip and the antibody chip multiple detection technology can meet the rapid and convenient, high-sensitivity and high-flux detection requirement of the mycotoxin in the agricultural product, and provides the technical support for the establishment of the mycotoxin pollution monitoring system in the agricultural products, the food and the feed, And also provides reference and strategy for the detection of other small molecular analytes.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S133
[Abstract]:Mycotoxin is a kind of small-molecule secondary metabolite produced by the fungus, and the crops are easy to be polluted by the mycotoxin in the aspects of growth, harvesting, storage and transportation, and more than 400 mycotoxins have been found, most of which are carcinogenic, The toxicity of teratogenic and mutagenic effects and mixed contamination is significantly enhanced. The mycotoxin can enter the food chain through the pollution of grain and feed or animal-derived food, and has serious harm to human and animal health, and has become the research focus in the field of food safety in recent years. The existing detection methods of mycotoxins include the biological identification method, the chemical analysis method, the instrument analysis method and the immunoassay method, and the like. The biological identification method is not strong in specificity, the chemical analysis method is low in precision, the analytical sensitivity of the instrument is high, the accuracy can be accurately quantified, the pretreatment of the sample is complicated and the detection instrument is expensive, and the use is difficult to be popularized in the basic-layer laboratory. The immune analysis method is rapid, high in sensitivity, good in specificity and low in cost, and has developed rapidly in the field of mycotoxin detection in recent years. Aflatoxin B1 (AFB1), aspergillotoxin A (OTA), fumonisin B1 (FB1) and Zealenone (ZEN), which are common and harmful to the agricultural products, are selected as the detection target, and the monoclonal antibody is used as the biological identification element. Based on the principle of competitive ELISA and immunochromatography, the rapid, sensitive and high-throughput immunodetection method of various mycotoxins is established by combining the techniques of signal amplification, immunosensing, antibody chip and the like. The main contents and results of this thesis are as follows:1. The immunological properties of the OTA monoclonal antibody 2D8 were identified by indirect competitive ELISA. The indirect competitive enzyme-linked immunosorbent assay (CI-ELISA) was established based on the antibody. The antibody 2D8 has a potency of 1: 1.024-106, a affinity constant Ka = 3.75-109L/ M, a heavy chain of an IgG1 type, a cross-reaction rate of 5.4% with an aspergiloxin B (Ochratoxin B, OTB), and AFB1, FB1, ZEN, Penicillium citrinin (CIT), Paecilomyces (Patulin, PAT), and a vomiting toxin (Deoxynivalenol, DON) No cross-reaction. The detection limit of ci-ELISA is 0.08 ng/ mL, the detection range is 0.12-1.18 ng/ mL, the recovery rate of the spike can reach 91.2-110.3%, the correlation with the commercial ELISA kit is good, the quantitative detection and analysis of the OTA in the grain and the feed sample can be used, and the foundation for the establishment of the subsequent other immunodetection methods is also provided. the method is based on the indirect competitive ELISA of the immunomagnetic beads and the biotin-streptavidin to quantitatively detect the corn trichoderma ketene to shorten the reaction time, improve the detection sensitivity, coat the ZEN monoclonal antibody as a solid phase carrier by using the nano magnetic beads as a solid phase carrier, and based on the principle of the competition reaction, A new enzyme-linked immunosorbent assay (MNP-bsELISA) was established using a biotin-streptavidin system. The detection limit of the method is 0.04 ng/ mL, the detection range is 0.07-2.41 ng/ mL, the recovery of the spike is 92.8-111.9%, the coefficient of variation is less than 10%, and the quantitative result of ZEN in the natural sample has a good correlation with the LC-MS/ MS method. The immune competitive reaction of the magnetic bead as the solid-phase carrier is optimized by using the magnetic bead-electrochemical immunosensing detection technology of the aspergillosis toxin A. The best mode is selected and the electrochemical signal detection technology is combined to establish the OTA electrochemical immunosensing detection technology. The detection limit of the method is 0.007 ng/ mL, the detection range is 0.01-0.82 ng/ mL, the recovery rate of the spike is 78.7-91.6%, the standard deviation and the coefficient of variation are all less than 15%, the stability is good, and the trace detection of the OTA in the sample can be realized. Based on the principle of immunochromatography, an immunochromatographic method for the detection of OTA and ZEN in grain and feed was established based on the principle of immunochromatography. The detection limit for OTA and ZEN is 0.625 ng/ mL and 1.25 ng/ mL respectively. The detection limit of the two-linked qualitative colloidal gold immunochromatographic test strip to OTA is 0.25 ng/ mL, the detection interval is 0.32-12.16 ng/ mL, the detection limit to ZEN is 0.5 ng/ mL, and the detection interval is 0.58-39.72 ng/ mL; The recovery of two mycotoxins in corn, wheat and feed samples was 79.6-105.2%. The detection results of the prepared two-linked immunochromatographic test strip and the LC-MS/ MS are better than that of the LC-MS/ MS, and the rapid qualitative detection and quantitative analysis of OTA and ZEN in the samples can be respectively met. the high-flux antibody chip quantitative technical mycotoxin mixed pollution condition of the four mycotoxins is detected at the same time, and the toxicity is obviously enhanced, and the high-flux antibody chip quantitative detection is carried out on the four common mycotoxins (AFB1, OTA, FB1 and ZEN) in the sample, Taking the nitrocellulose membrane as a substrate, and using biotin. And the streptavidin system is used for establishing an antibody chip detection mode. The detection limits of AFB1, OTA, FB1 and ZEN were 0.21, 0.19, 0.24 and 0.09 ng/ mL, respectively. The detection interval was 0.47-55.69, 0.48-127.11, 0.56-92.57 and 0.22-31.36 ng/ mL, respectively. The recovery rate of the actual sample is 79.2-113.4%, the coefficient of variation is less than 10%, the stability is good, and the mycotoxin can be tested for multiple quantitative detection, and has wide application prospect in the field of the mixed pollution monitoring of the mycotoxin. in conclusion, that present study combine the specific monoclonal antibody of the mycotoxin with the signal amplification and high-flux detection system of the magnetic nano-material, the biotin-streptavidin, the electrochemistry, the chip and the like, and respectively establish the ci-ELISA, the MNP-bsELISA and the magnetic bead electrochemical immunosensor, the two-way qualitative and quantitative colloidal gold immunochromatographic test strip and the antibody chip multiple detection technology can meet the rapid and convenient, high-sensitivity and high-flux detection requirement of the mycotoxin in the agricultural product, and provides the technical support for the establishment of the mycotoxin pollution monitoring system in the agricultural products, the food and the feed, And also provides reference and strategy for the detection of other small molecular analytes.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S133
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5 王t，
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