基于限制性核酸內(nèi)切酶放大技術(shù)的沙門氏菌檢測生物傳感器
發(fā)布時間：2018-09-03 11:16
【摘要】：本文詳細介紹了食源性致病菌的危害、常見種類及研究現(xiàn)狀。核酸適配體的功能及其特點和應(yīng)用。限制性核酸內(nèi)切酶放大技術(shù)的特點及其在生物傳感器檢測中的應(yīng)用。并對核酸適配體生物傳感器做了相關(guān)改進,用于檢測食源性致病菌。1、我們通過適配體的特異性識別和吸附功能設(shè)計了簡便、省時、一步檢測鼠傷寒沙門氏菌的熒光生物傳感器。該方案主要基于鏈置換放大-聚合酶延伸對目標物進行靈敏檢測。實驗巧妙設(shè)計了一個拱形探針(由aptamer和primer構(gòu)成)和一個發(fā)夾探針(HP)。發(fā)夾的3’端和5’端分別修飾了熒光基團和猝滅基團。只有當(dāng)目標物沙門氏鼠傷寒菌存在的條件下,才能夠破壞拱形探針,釋放primer鏈。被釋放的primer鏈打開發(fā)夾探針,使猝滅基團和熒光基團遠離,產(chǎn)生熒光。同時在聚合酶的延伸作用下,發(fā)生連置換反應(yīng),使primer鏈能夠重復(fù)利用,達到信號放大的目的。因為信號放大和熒光強度檢測的本質(zhì)上的高靈敏性,600 cfu m L-1的致病菌可以在2 h內(nèi)得到檢測,這樣比之前報道的檢測方法更加的敏感和有效。因此它為發(fā)展鏈置換放大效應(yīng)基于超靈熒光強度來檢測致病菌的發(fā)展和食品安全分析提供了良好的應(yīng)用平臺。2、在這項工作中,基于目標物誘導(dǎo)的指數(shù)放大反應(yīng),我們發(fā)明了一種簡單、迅速、等溫、和超靈敏的均勻比色適配體傳感器來對致病菌進行探測。這種適配體-引物探針包括結(jié)合鼠傷寒沙門氏菌的適配體和一段引物序列,這種發(fā)夾探針包括一個引物單元,這個適配體-引物探針被用于識別目標物并且通過基于目標物而激發(fā)指數(shù)放大反應(yīng)(EXPAR)。由于EXPAR耦合DNA酶的放大策略,在目標物致病菌出現(xiàn)的時候,將會在溶液中形成大量大G-四聯(lián)體低聚物,然后在鉀離子和血紅素的輔助下,又折疊成為G-四聯(lián)體/血紅素復(fù)合物,這種復(fù)合物對于過氧化氫有很強的催化活性,并且產(chǎn)生很強的紫外吸收。這種新穎的EXPAR耦合模仿過氧化酶催化放大技術(shù)通過比色法來探測致病菌。在最佳條件下,這種生物傳感器對于致病菌的檢測是非常靈敏的,在4 h之內(nèi)它的檢測限度是80 cfu m L-1.同時,我們的生物傳感器對于目標物致病菌的檢測有很高的專一性,同時,還有檢測快速、廉價、操作簡單、不需要進行標記和加入不穩(wěn)定試劑的優(yōu)點。因此,這種基于EXPAR耦合模擬DNA酶的比色方法將會成為檢測致病菌的有效和實用的平臺,其在食品安全分析和環(huán)境監(jiān)測上都有很大的用處。
[Abstract]:In this paper, the harm, common species and research status of foodborne pathogenic bacteria are introduced in detail. Functions, characteristics and applications of aptamers of nucleic acids. The characteristics of restriction endonuclease amplification and its application in biosensor detection. We also improved the aptamer biosensor for the detection of foodborne pathogenic bacteria. We designed a simple and time-saving fluorescent biosensor for the detection of Salmonella typhimurium through the specific recognition and adsorption function of the aptamer. This scheme is mainly based on chain replacement amplification-polymerase extension for sensitive detection of the target. An arched probe (composed of aptamer and primer) and a hairpin probe, (HP)., have been ingeniously designed. The 3 'and 5' ends of the hairpin were modified with fluorescence group and quenching group, respectively. Only in the presence of Salmonella typhimurium could the arched probe be destroyed and the primer chain released. The released primer chain opens the hairpin probe, leaving the quenching group and the fluorescence group away to produce fluorescence. At the same time, under the extension of polymerase, the primer chain can be reused to achieve the purpose of signal amplification. Because of the intrinsic sensitivity of signal amplification and fluorescence intensity detection, the pathogens of 600 cfu mL ~ (-1) can be detected within 2 h, which is more sensitive and effective than the previously reported detection method. Therefore, it provides a good application platform for the development of chain replacement amplification based on superfluorescence intensity to detect pathogenic bacteria and food safety analysis. We have developed a simple, rapid, isothermal, and hypersensitive uniform colorimetric aptamer sensor to detect pathogens. The aptamer primer probe consists of an aptamer bound to Salmonella typhimurium and a primer sequence, and the hairpin probe includes a primer unit. This aptamer primer probe is used to identify the target and to excite the exponential amplification reaction based on the target (EXPAR). Due to the amplification strategy of EXPAR coupled with DNA enzyme, a large number of large G- tetraad oligomers will be formed in the solution when the target pathogen appears, and then folded into G- quadruplex / heme complex with the help of potassium ion and heme. The complex has strong catalytic activity for hydrogen peroxide and strong UV absorption. The novel EXPAR coupled imitating peroxidase catalytic amplification technique was used to detect pathogenic bacteria by colorimetry. Under the optimum conditions, the biosensor is very sensitive to the detection of pathogenic bacteria, and the detection limit is 80 cfu ml -1 within 4 h. At the same time, our biosensors have the advantages of high specificity for the detection of target pathogens, rapid detection, low cost, simple operation, and no need to label and add unstable reagents. Therefore, this colorimetric method based on EXPAR coupled simulation of DNA enzyme will be an effective and practical platform for the detection of pathogenic bacteria, and it will be very useful in food safety analysis and environmental monitoring.
【學(xué)位授予單位】：濟南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：TS207.4;TP212.3
本文編號：2219823
[Abstract]:In this paper, the harm, common species and research status of foodborne pathogenic bacteria are introduced in detail. Functions, characteristics and applications of aptamers of nucleic acids. The characteristics of restriction endonuclease amplification and its application in biosensor detection. We also improved the aptamer biosensor for the detection of foodborne pathogenic bacteria. We designed a simple and time-saving fluorescent biosensor for the detection of Salmonella typhimurium through the specific recognition and adsorption function of the aptamer. This scheme is mainly based on chain replacement amplification-polymerase extension for sensitive detection of the target. An arched probe (composed of aptamer and primer) and a hairpin probe, (HP)., have been ingeniously designed. The 3 'and 5' ends of the hairpin were modified with fluorescence group and quenching group, respectively. Only in the presence of Salmonella typhimurium could the arched probe be destroyed and the primer chain released. The released primer chain opens the hairpin probe, leaving the quenching group and the fluorescence group away to produce fluorescence. At the same time, under the extension of polymerase, the primer chain can be reused to achieve the purpose of signal amplification. Because of the intrinsic sensitivity of signal amplification and fluorescence intensity detection, the pathogens of 600 cfu mL ~ (-1) can be detected within 2 h, which is more sensitive and effective than the previously reported detection method. Therefore, it provides a good application platform for the development of chain replacement amplification based on superfluorescence intensity to detect pathogenic bacteria and food safety analysis. We have developed a simple, rapid, isothermal, and hypersensitive uniform colorimetric aptamer sensor to detect pathogens. The aptamer primer probe consists of an aptamer bound to Salmonella typhimurium and a primer sequence, and the hairpin probe includes a primer unit. This aptamer primer probe is used to identify the target and to excite the exponential amplification reaction based on the target (EXPAR). Due to the amplification strategy of EXPAR coupled with DNA enzyme, a large number of large G- tetraad oligomers will be formed in the solution when the target pathogen appears, and then folded into G- quadruplex / heme complex with the help of potassium ion and heme. The complex has strong catalytic activity for hydrogen peroxide and strong UV absorption. The novel EXPAR coupled imitating peroxidase catalytic amplification technique was used to detect pathogenic bacteria by colorimetry. Under the optimum conditions, the biosensor is very sensitive to the detection of pathogenic bacteria, and the detection limit is 80 cfu ml -1 within 4 h. At the same time, our biosensors have the advantages of high specificity for the detection of target pathogens, rapid detection, low cost, simple operation, and no need to label and add unstable reagents. Therefore, this colorimetric method based on EXPAR coupled simulation of DNA enzyme will be an effective and practical platform for the detection of pathogenic bacteria, and it will be very useful in food safety analysis and environmental monitoring.
【學(xué)位授予單位】：濟南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：TS207.4;TP212.3
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，本文編號：2219823
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