本文選題：單分子核酸擴(kuò)增 + 數(shù)字PCR�。� 參考：《浙江大學(xué)》2013年博士論文

【摘要】：數(shù)字PCR,以其能通過(guò)單分子擴(kuò)增實(shí)現(xiàn)核酸拷貝數(shù)絕對(duì)定量被稱(chēng)為第三代PCR技術(shù)。特別是以微流控芯片為基礎(chǔ)的數(shù)字PCR方法,因其超高的靈敏度、準(zhǔn)確度及低試劑消耗等優(yōu)點(diǎn)而得到迅速發(fā)展。然而現(xiàn)有的芯片型和液滴型數(shù)字PCR平臺(tái)操作復(fù)雜費(fèi)時(shí),需要許多外接管路和配套控制系統(tǒng),并未完全發(fā)揮微流控芯片的優(yōu)勢(shì),由此商品化的儀器價(jià)格昂貴且全部依賴(lài)進(jìn)口,制約了數(shù)字PCR技術(shù)在我國(guó)生命科學(xué)技術(shù)領(lǐng)域的應(yīng)用和普及。本論文在總結(jié)數(shù)字PCR原理、方法及學(xué)習(xí)研制基于氣動(dòng)微閥大規(guī)模集成流路數(shù)字PCR芯片技術(shù)的基礎(chǔ)上,優(yōu)化了納升以下體積的單分子PCR反應(yīng)的反應(yīng)體系與反應(yīng)條件,在此基礎(chǔ)上取得了如下創(chuàng)新性結(jié)果：1)首次成功研制具有完全自主知識(shí)產(chǎn)權(quán)的自吸分液式集成流路(Integrated Fluidic Circuits, IFC)數(shù)字PCR芯片。該芯片利用PDMS材料的儲(chǔ)氣特性和緩慢的氣體分子擴(kuò)散過(guò)程,將負(fù)壓預(yù)先儲(chǔ)存在PDMS芯片內(nèi)為芯片進(jìn)樣提供動(dòng)力源,芯片由與由主通道相連接的微反應(yīng)室陣列組成,利用油水兩相不相容的性質(zhì)以及兩相之間的界面張力,連續(xù)的將PCR反應(yīng)液和油相吸入芯片,實(shí)現(xiàn)樣品的隨機(jī)分配和隔離。該芯片無(wú)動(dòng)力源,以油相代替微閥,實(shí)現(xiàn)了芯片的整合集成化。2)芯片內(nèi)部采用一種新的氟硅烷納米涂層,厚度僅10 nm就能有效的防治反應(yīng)樣品的蒸發(fā),與已有的parylene C防水涂層相比,操作簡(jiǎn)單,無(wú)需活化,成本低廉,具有無(wú)可比擬的優(yōu)越性。油相可以在加熱時(shí)固化,防止樣品溶液在高溫時(shí)對(duì)流,避免了反應(yīng)的交叉污染。3)設(shè)計(jì)了密度為1156反應(yīng)/cm2的數(shù)字PCR芯片,尺寸與與蓋玻片(50 mm × 24 mm× 4 mm)大小相當(dāng),分為四個(gè)檢測(cè)區(qū)域,每個(gè)區(qū)域包含1280個(gè)小室,小室尺寸150 pm × 150 pm × 250μm,體積為6.5 nL,可同時(shí)檢測(cè)四個(gè)樣品或者同一樣品的四個(gè)濃度。應(yīng)用該芯片分別進(jìn)行了數(shù)字環(huán)介導(dǎo)等溫?cái)U(kuò)增反應(yīng)(digital LAMP)和數(shù)字PCR反應(yīng)(digital PCR),對(duì)芯片的靈敏度、準(zhǔn)確度、測(cè)量誤差進(jìn)行了驗(yàn)證,建立了該芯片的數(shù)據(jù)統(tǒng)計(jì)方法,成功對(duì)人類(lèi)持家基因和腫瘤相關(guān)基因進(jìn)行了精確定量。4)設(shè)計(jì)了密度為1562500反應(yīng)/cm2,總反應(yīng)小室個(gè)數(shù)達(dá)到6,400,000的數(shù)字LAMP芯片,每個(gè)區(qū)域包含1,600,000個(gè)小室,小室尺寸為4 μmx4μm×10μm,體積160飛升,反應(yīng)總體積僅僅1此,理論上檢測(cè)能力可達(dá)到7個(gè)數(shù)量級(jí),準(zhǔn)確度可達(dá)99%,該芯片的檢測(cè)能力大大超過(guò)了現(xiàn)有的數(shù)字PCR芯片,具有超高通量、低消耗、微型化、集成化和超大規(guī)模平行處理等方面的優(yōu)勢(shì),可用于現(xiàn)場(chǎng)即時(shí)檢測(cè)。5)基于蛋白質(zhì)定量PLA技術(shù),利用該芯片首次成功建立了蛋白質(zhì)單分子精確定量的數(shù)字親和連接反應(yīng)(digital PLA),對(duì)EGFR蛋白質(zhì)分子進(jìn)行單分子計(jì)數(shù)精確定量。與數(shù)字ELISA相比,該檢測(cè)方法更加簡(jiǎn)單,特異性、準(zhǔn)確度、靈敏性等更高,為臨床蛋白質(zhì)標(biāo)志物的檢測(cè)提供了新工具。綜上,該自吸分液式數(shù)字PCR芯片自動(dòng)進(jìn)樣,無(wú)需動(dòng)力驅(qū)動(dòng)及微閥門(mén)控制等系統(tǒng),消除了數(shù)字PCR芯片對(duì)外界控制系統(tǒng)的依賴(lài),使數(shù)字核酸擴(kuò)增芯片從操控芯片各個(gè)功能單元的儀器中獨(dú)立出來(lái),解決了芯片與外界系統(tǒng)的連接問(wèn)題,比現(xiàn)有的數(shù)字PCR平臺(tái)更加簡(jiǎn)單、實(shí)用。具有易于操作、敏感度高(單分子)、準(zhǔn)確性好(絕對(duì)定量)、節(jié)省樣品和試劑(數(shù)納升臌反應(yīng)室)、交叉污染小等特點(diǎn),適合于任何實(shí)驗(yàn)室使用。為解決生命科學(xué)領(lǐng)域中基因拷貝數(shù)精確定量難的關(guān)鍵問(wèn)題包括腫瘤早期診斷、非侵入性產(chǎn)前診斷、細(xì)菌和病毒檢測(cè)、單細(xì)胞基因組、單分子蛋白質(zhì)檢測(cè)等研究提供一種新儀器。
[Abstract]:Digital PCR is known as the third generation PCR technology with its single molecule amplification by single molecule amplification. In particular, the digital PCR method based on microfluidic chips has developed rapidly because of its high sensitivity, accuracy and low reagent consumption. However, some chip and drop digital PCR platforms operate complex. Many external pipelines and matching control systems are needed in the miscellaneous fee, and the advantages of microfluidic chips are not fully played. The commercialized instruments are expensive and all depend on import, which restricts the application and popularization of digital PCR technology in the field of life science and technology in our country. This paper summarizes the principle of digital PCR, methods and study and development based on gas. On the basis of large scale integrated flow digital PCR chip technology of dynamic micro valve, the reaction system and reaction conditions of the single molecule PCR reaction of the volume below the NL are optimized. On this basis, the following innovative results are obtained: 1) the self-priming liquid integrated flow path (Integrated Fluidic Circuits) with fully autonomous intellectual property rights is successfully developed for the first time. IFC) digital PCR chip. The chip uses the gas storage characteristics of the PDMS material and the slow gas molecular diffusion process, and stores the negative pressure in the PDMS chip in advance to provide the power source for the chip sample. The chip is composed of the microreaction chamber array connected with the main channel, using the incompatible properties of the two phase of the oil and water and the interfacial tension between the two phases. Continuous PCR reacting liquid and oil phase are inhaled to realize the random distribution and isolation of the samples. The chip has no power source, the oil phase is replaced by the micro valve and the integrated integrated.2 of the chip is implemented. A new fluorossilane nano coating is used inside the chip. The thickness of only 10 nm can effectively prevent the evaporation of the reaction samples from the existing parylene C. Compared with the water coating, the operation is simple, no activation, low cost and incomparable superiority. The oil phase can be cured at heating, prevent the sample solution from convection in high temperature and avoid the cross contamination.3 of the reaction. The digital PCR chip with a density of 1156 reaction /cm2 is designed, and the size is equal to the size of the cover glass (50 mm * 24 mm * 4 mm). For four detection regions, each area contains 1280 chambers, the size of the chamber is 150 PM * 150 PM x 250 mu m, and the volume is 6.5 nL, and four samples or four concentrations of the same sample can be detected simultaneously. The chip is used for the digital ring mediated isothermal amplification reaction (digital LAMP) and the digital PCR reaction (digital PCR), and the sensitivity of the chip to the chip. The degree, accuracy, measurement error was verified, and the data statistics method of the chip was established. The accurate quantitative.4 of the human family and tumor related genes was successfully designed. The digital LAMP chip with a density of 1562500 reaction /cm2 and the total number of small cells with a total number of 6400000 had 1600000 small chambers in each area and the size of the chamber was 4. MX4 mu m x 10 mu m, the volume 160 fly up, the total volume of the reaction is only 1. In theory, the detection ability can reach 7 orders of magnitude and the accuracy is up to 99%. The detection ability of the chip is much more than the existing digital PCR chip. It has the advantages of super high throughput, low consumption, miniaturization, integration and large scale parallel processing. Time detection.5) based on the protein quantitative PLA technology, the digital affinity connection reaction (digital PLA) of the protein single molecule was established for the first time, and the single molecule count of EGFR protein molecules was quantified. Compared with the digital ELISA, the detection method was more simple, more specific, more accurate, and more sensitive, and so on. The detection of clinical protein markers provides a new tool. To sum up, the automatic sampling of the self absorption digital PCR chip, without power drive and micro valve control system, eliminates the dependence of the digital PCR chip on the external control system, and makes the digital nucleic acid amplification chip independent from the instrument of each functional unit of the control core and solves the problem. The connection between the chip and the external system is simpler and more practical than the existing digital PCR platform. It has the characteristics of easy operation, high sensitivity (single molecule), good accuracy (absolute quantification), saving of samples and reagents (number of liters and heave reaction rooms), small cross contamination and so on. It is suitable for the use of any laboratory in the life science field. The key problems of precision and quantification of the number of shells include early diagnosis of tumor, non-invasive prenatal diagnosis, bacterial and viral detection, single cell genome, single molecule protein detection, and other research instruments.
【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】：2013
【分類(lèi)號(hào)】：R440
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本文編號(hào)：1972585