【作者】 王星宇；

【导师】 梁兴国；

【作者基本信息】 中国海洋大学 ， 食品科学， 2014， 博士

【摘要】 核酸扩增是一项基本的生物技术，在医疗、农业、环境监测和取证等各个方面有着不可替代的作用。传统的核酸扩增使用聚合酶链式反应，即PCR技术。作为一种经典的扩增检测方法，同以其为基础发展而来的实时荧光定量PCR（qPCR）技术，被广泛的应用于核酸的扩增与检测领域。但这一技术在食品领域应用时会出现易受污染产生假阳性，有聚合酶抑制剂时使用困难，背景核酸大量存在时灵敏度大幅降低等问题。这些缺点限制了PCR直接用于检测食品中外源微生物。同时，作为一种变温扩增技术，PCR特异性的发挥需要精确的控温仪器，不符合基层现场快速检测的要求。因此需要开发一种新的核酸扩增技术，能够摆脱对精确控温仪器的依赖，同时能够从食品中检测出外源病毒和细菌，即能够在大量背景核酸存在的条件下特异性检测目标序列的存在，以达到在食品领域真正广泛应用的目标。滚环扩增（Rolling circle amplification，RCA），是一种在等温条件下就能特异性扩增目标片段的扩增技术，主要利用环状模板和具有链置换能力的DNA聚合酶环绕模板进行周期性的复制。常见技术应用为都以锁式探针RCA（padlock-RCA）引发扩增。虽然灵敏度高，但也有单链探针不稳定、探针与模板容易错配、扩增产物并非目标序列而是互补的探针自身的缺点。针对于此，本文使用了一种全新的目标成环RCA（Target-circularization RCA，TC-RCA）：利用限制性内切酶酶切样品，产生具有9nt粘性末端的序列；其次，设计与目标序列粘性末端完全互补配对的双链接头，在连接酶的作用下同目标序列连接成环；利用具有链置换活性的DNA聚合酶，在两条引物的作用下引发超分支-RCA（Hyper-branched RCA）；最后，扩增产物再经过限制性内切酶酶切，得到扩增后的目的片段。在此基础上，利用高特异性的DNA连接酶、生物素修饰接头、链霉亲和素磁珠以及磁性分离等手段，构建了磁珠辅助的TC-RCA技术。该项技术能够在等温条件下从大量背景核酸中检测出目标序列，从根本上解决了检测特异性和灵敏度之间的矛盾关系，特别适用于检测食品中有害微生物的存在。论文主要从以下几个方面研究建立特异性核酸检测方法。（1）TC-RCA的可行性，证明了TC-RCA具有在无背景核酸条件下扩增目标DNA片段的能力，敏感度为6×104个拷贝分子。同时，接头能稳定结合于固相磁珠表面，并且设计的接头固定于固相表面后不影响连接与扩增，为TC-RCA在芯片上实现目标成环等温扩增提供了技术支持和理论依据。（2）TC-RCA可以在大量背景核酸中检测出目标序列，敏感度达到6×106个拷贝分子，但结果伴有非特异性扩增。使用连接忠实度较高的Taq DNA连接酶进行连接，没有能够达到提高特异性的目的。主要是由于Taq DNA连接酶在非正常的缓冲液条件下，区别错配能力降低所致。说明TC-RCA已经初步具备成为一种新型检测方法的能力，同时为后续的实验提供了解决问题的思路和依据。（3）通过磁珠辅助将扩增子与背景DNA和Taq DNA连接酶分离，同时结合其它条件的优化探索，使得磁珠辅助的TC-RCA可以在高背景核酸存在的条件下特异性地扩增目的片段，敏感度为60个拷贝分子，并且没有任何非特异性扩增出现。经过对各个步骤的精简优化，检测结果24h之内即可获得。磁珠辅助的TC-RCA通用性强，更适用于基层和现场快速检测。（4）磁珠辅助TC-RCA适用于检测水产品中有害细菌，SYBR Green I染色结果表明该技术可以特异地检测出水产品中的外源细菌，最低检测限达到100个细菌基因序列拷贝分子。同其它检测水产品中有害微生物的方法相比，本方法不需要进行增菌实验，检测的结果可以直接反应食品被细菌污染的最初状态。同时反应不依赖精密控温仪器，常用水浴锅就满足检测要求，为基层现场快速检测食品中细菌提供了一种强有力的技术手段。更多还原

【Abstract】 Nucleic acid amplification is a basic biotechnology that plays an irreplaceablerole in all aspects such as health care, agriculture development, environmentalprotection and forensics analysis. As a classical amplification technology, PCR withits evolved fashion named real-time quantitative PCR (RT-PCR), are widely used inthose fields. However, vulnerability to the contamination that generates false positives,inhibition caused by polymerase inhibitor, impaired sensitivity delivered by massivebackground nucleic acid in, discrediting PCR as a powerful technology for thedetection of exogenous microbes in food. Meanwhile, as a non-thermal amplificationtechnique, PCR relies on precise thermostable equipments, which does not meet therequirements of the on-spot-rapid detection. Therefore, the great demand for anoriginal amplification technology that is free from expensive equipment and immuneto the interference from background nucleic acids, which can be applied for foodsecurity administration, is enhancedAs an isothermal detection method for overcoming the shortcomings of PCR,RCA (rolling circle amplification) has gained a great attention over the past decade.For the feasibility and sensitivity, padlock-RCA has been investigated in many labors.The specificity of padlock-RCA is based on the specific hybridization of two ends of apre-circular DNA (probe) with the target sequence. Nonetheless, even in the case thatseveral mismatches are present at the position not close to the ligation site, the probecan also be circularized and end up with false positive results. Moreover, theamplification reaction of padlock-RCA only generates tandem copies of the addedprobe itself but not the DNA target.Here we present a novel DNA detection method, termed “Target-CircularizationRolling Circle Amplification”(TC-RCA) based on rolling circle amplification, Adigestion step is employed to generate a number of shorter fragments including theDNA target with9nt sticky ends. Then a ligase is utilized to ligate the adaptor and the target DNA form a double stranded circle with a gap. Phi29DNA polymerase is thenused to amplify the circle by RCA using the integral strand as the template and theopen strand as the primer. The hyper-branched RCA is achieved with the help of twoprimers complementary to the target sequence. Finally, the products consisting oftandem copies of dsDNA target were digested by restriction endonuclease (TspR I) atthe cutting site as5’-NNCASTGNN-3’. Hence, the target DNA meant to be replicatedor detected is obtained with the augmentation of several magnitudes. Based onTC-RCA, several strategies such as high fidelity DNA ligase, biotinylated adaptor,streptavidin magnetic bead (SMB) and magnetic separation have been taken toimprove the sensitivity and specificity of TC-RCA to be an isothermal amplificationtechnology for detection harmful bacteria in food product. The following gists havebeen studied in this paper.1) The feasibility of TC-RCA has been proved. TC-RCA is capable of amplifyingat least6×104copies of target molecule in the absence of background DNA. Besides,the adaptor can be immobilized on solid phase surface of magnetic beads, andimmobilization does not affect the ligation and afterward amplification. This providestechnical support and theoretical basis for TC-RCA based on chip isothermalamplification.2) Detection of the target sequence in the presence of background DNA isachieved by TC-RCA. The detection limit is6×106copies of target molecule alongwith nonspecific amplifications. Taq DNA ligase featuring high ligation fidelity failsto promote the detection specificity, since Taq DNA ligase tolerates mismatches underabnormal conditions. This indicates that TC-RCA can be used for primary detection infood security control and intrigues us to contrive for new solutions for the problems.3) By utilizing SMB that realizes the separation between circular amplicon andinhibitors such as Taq DNA ligase and background DNA, SMB-assisted TC-RCAupgrade to a level that can detect at least60copies of target sequence in the presenceof massive background DNA. The desirable specificity is accomplished for nounexpected amplification occurred. With effort towards efficiency improvement, thedetection result can be obtained with24h, which paves the way for SMB-assisted TC-RCA to a general-use, rapid and precise detection protocol.4) SMB-assisted TC-RCA is proved to be applicable to detect harmful bacteriafrom aquatic food under isothermal conditions with great sensitivity and specificity.SYBR Green I displays the same results with electrophoresis. Comparing to PCR andother protocols in food control, this strategy can be conducted away from enrichmentculture, so the original infection status is allowed to investigate. In addition, ourprotocol avoids complicated thermal cycling program and expensive equipment, andheating in a period of time using a simple water incubator is sufficient to amplifyDNA to detectable levels. SMB-assisted TC-RCA is considered to be promoted as afast, rapid, real-time detection strategy for detecting harmful microbes in food andrelated products.更多还原