本文選題：沙門氏菌 + MicroRNA　； 參考：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：核酸作為一類最基本的生命物質(zhì),在遺傳信息處理過程中具有至關(guān)重要的作用,包括遺傳信息的存儲(chǔ)、復(fù)制和傳遞等,因此成為生命科學(xué)中廣泛研究的熱點(diǎn)之一。作為一類生物標(biāo)志物,核酸可能在一些疾病(例如癌癥)的發(fā)生和發(fā)展過程中,提供了一些很有價(jià)值的信息。尤其是在一些疾病的早期,這些相關(guān)核酸生物標(biāo)志物的濃度處于很低的水平。因此,建立快速、靈敏和特異的方法來檢測(cè)這些低豐度的疾病相關(guān)核酸標(biāo)志物,對(duì)于基因治療、突變分析和臨床診斷具有重要意義。表面等離子共振傳感器是20世紀(jì)90年代提出的一種用于生物分子間相互作用分析(BIA)的親和型生物傳感技術(shù)。近年來,由于該技術(shù)具有免標(biāo)記、實(shí)時(shí)監(jiān)測(cè)、耗樣量少和靈敏度高等特點(diǎn),受到了相關(guān)領(lǐng)域研究人員的極大的關(guān)注,并廣泛應(yīng)用于生物標(biāo)志物的分析。本研究以表面等離子共振生物傳感器為檢測(cè)平臺(tái),結(jié)合生物分析化學(xué)和分子生物學(xué)相關(guān)技術(shù),提出了簡(jiǎn)便靈敏的核酸檢測(cè)策略。本論文主要包括以下兩個(gè)部分：1.親和素適體放大的表面等離子共振傳感器檢測(cè)沙門氏菌invA基因基于表面等離子共振傳感芯片,整合了信號(hào)放大元件親和素適體和修飾的不對(duì)稱PCR反應(yīng),建立了一種檢測(cè)沙門氏菌的高靈敏方法。首先在傳感芯片表面修飾巰基修飾的探針,然后加入靶物質(zhì)和親和素適體形成三明治雜交結(jié)構(gòu)。當(dāng)親和素加入體系后,親和素適體可與親和素形成復(fù)合體,從而實(shí)現(xiàn)信號(hào)的放大。在最優(yōu)的條件下,該方法檢測(cè)invA基因的線性范圍是50 pM到200 nM,最低檢測(cè)限為20 pM。該策略成功地檢測(cè)到低達(dá)60 CFU mL-1的沙門氏菌。所建立的方法靈敏度高、選擇性強(qiáng)、穩(wěn)定性好,這些優(yōu)點(diǎn)使得該策略在食物、臨床和環(huán)境樣本中對(duì)沙門氏菌的篩查具有很好的應(yīng)用前景。2.基于表面等離子共振傳感器的新型DNA納米技術(shù)檢測(cè)microRNAMicroRNA (miRNA)在許多疾病發(fā)生和發(fā)展中具有十分重要的調(diào)節(jié)作用,已成為一類很有前景的疾病生物標(biāo)志物。本研究基于表面等離子共振生物傳感器平臺(tái),結(jié)合錯(cuò)配型的催化莖環(huán)自組裝(CHA)和鏈霉親和素適體,構(gòu)建了一種放大策略,用于靶miRNA高靈敏特異的檢測(cè)。在靶miRNA的存在下,可觸發(fā)CHA放大反應(yīng),從而形成靶miRNA的循環(huán)使用并產(chǎn)生大量的CHA反應(yīng)產(chǎn)物。另外,大量的CHA反應(yīng)產(chǎn)物一端可與修飾在裸金芯片表面修飾成功的捕獲探針雜交結(jié)合,另一端則可耦合鏈霉親和素,完成檢測(cè)信號(hào)的放大和輸出。由于無(wú)酶的CHA放大反應(yīng)和免標(biāo)記SPR生物傳感器的優(yōu)良性能,在最優(yōu)的實(shí)驗(yàn)條件時(shí),該方法能夠檢測(cè)靶miRNA的線性范圍為5 pM到100nM,最低檢測(cè)限可達(dá)到1 pM,相關(guān)系數(shù)為0.9968,并且具有很好的特異性。綜上所述,基于SPR的生物傳感策略很有潛力發(fā)展成為一種用于miRNA檢測(cè)的新型手段,以更好地用于醫(yī)學(xué)研究和臨床早期診斷。
[Abstract]:Nucleic acid, as the most basic life substance, plays an important role in the process of genetic information processing, including the storage, replication and transmission of genetic information, so it has become one of the hot spots in life science. As a kind of biomarker, nucleic acid may provide some valuable information in the occurrence and development of some diseases (such as cancer). Especially in the early stages of some diseases, the concentration of these related nucleic acid biomarkers is very low. Therefore, it is important for gene therapy, mutation analysis and clinical diagnosis to establish a rapid, sensitive and specific method for the detection of these low-abundance disease-related nucleic acid markers. Surface plasmon resonance sensor is a kind of affinity biosensor technology proposed in 1990s for biomolecular interaction analysis (BIA). In recent years, the technology has been paid great attention by researchers in related fields because of its characteristics such as no marking, real-time monitoring, small sample consumption and high sensitivity, and has been widely used in the analysis of biomarkers. In this study, a simple and sensitive nucleic acid detection strategy was proposed on the basis of surface plasmon resonance biosensor and related techniques of bioanalytical chemistry and molecular biology. This thesis mainly includes the following two parts: 1. Detection of Salmonella invA Gene based on Surface Plasmon Resonance Sensor Chip, the affinin-amplified aptamer and modified asymmetric PCR reaction are integrated. A highly sensitive method for the detection of salmonella was established. First, the thiol modified probe was modified on the surface of the sensor chip, and then the target material and the aptamer were added to form the sandwich hybrid structure. When affin is added into the system, the aptamer can form a complex with the aptamer, which can amplify the signal. Under the optimal conditions, the linear range of detection of invA gene by this method is from 50 pm to 200 nm, and the minimum detection limit is 20 pm. The strategy successfully detected salmonella as low as 60 CFU mL-1. The established method has the advantages of high sensitivity, high selectivity and good stability, which makes the strategy has a good application prospect in food, clinical and environmental samples for Salmonella screening. A novel DNA nanotechnology based on surface plasmon resonance (SSRR) has played an important role in regulating the occurrence and development of many diseases and has become a promising biomarker of disease. Based on the surface plasmon resonance biosensor platform and the mismatched catalytic stem ring self-assembly (cha) and streptavidin aptamer, an amplification strategy was constructed for the detection of target miRNA with high sensitivity and specificity. In the presence of target miRNA, the CHA amplification reaction can be triggered, resulting in the recycling of target miRNA and the production of a large number of CHA reaction products. In addition, a large number of CHA products can be hybridized with the successfully modified capture probe modified on the surface of bare gold chip, and the other end can be coupled with streptavidin to amplify and output the detection signal. Due to the excellent performance of non-enzymatic CHA amplification reaction and labeled free SPR biosensor, under the optimal experimental conditions, This method can detect target miRNA in the linear range from 5 pm to 100 nm, the minimum detection limit can reach 1 pm, the correlation coefficient is 0. 9968, and it has good specificity. In conclusion, the biosensor strategy based on SPR has the potential to develop into a new method for miRNA detection, which can be better used in medical research and early clinical diagnosis.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.6

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