本文關(guān)鍵詞： 等溫?cái)U(kuò)增 信號(hào)放大 分子信標(biāo) 核酸檢測(cè) POCT　出處：《青島科技大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：核酸作為重要的遺傳信息生物分子,其檢測(cè)被廣泛的應(yīng)用在食品安全、生物醫(yī)學(xué)檢測(cè)、環(huán)境檢測(cè)等諸多領(lǐng)域。聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase Chain Reaction,PCR)是目前應(yīng)用最廣泛的核酸擴(kuò)增檢測(cè)方法之一,然而PCR技術(shù)需要專(zhuān)門(mén)的熱循環(huán)儀,限制了其在疾病的即時(shí)診斷等領(lǐng)域的應(yīng)用。因此,發(fā)展更靈敏、快速的核酸等溫檢測(cè)方法具有重要意義。本論文構(gòu)建了幾種簡(jiǎn)單、快速的核酸等溫?cái)U(kuò)增檢測(cè)新方法,分別對(duì)DNA和RNA進(jìn)行了快速、靈敏和特異地檢測(cè)。在第二章中,本論文建立了一種基于雙切口分子信標(biāo)的等溫?cái)U(kuò)增檢測(cè)DNA新方法的研究。用該方法對(duì)提取的大腸桿菌16S r DNA進(jìn)行實(shí)時(shí)熒光檢測(cè),當(dāng)檢測(cè)靶標(biāo)的量在100 fmol到10 amol之間時(shí)呈良好的線(xiàn)性,最低檢測(cè)限為10 amol;通過(guò)在引物上設(shè)計(jì)突變堿基進(jìn)行檢測(cè),驗(yàn)證了該方法具有較強(qiáng)的特異性;并用該方法在復(fù)雜體系中檢測(cè)時(shí)呈現(xiàn)較強(qiáng)的抗干擾能力。此外,它是一鍋式的等溫鏈置換擴(kuò)增方法,不需要額外的操作步驟,極大的簡(jiǎn)化了實(shí)驗(yàn)步驟,并降低了樣品污染的可能性。它的這些優(yōu)點(diǎn)使該方法在核酸的現(xiàn)場(chǎng)快速檢測(cè)及即時(shí)檢測(cè)(Point-of-care testing,POCT)中更具有實(shí)用性。目前的RNA檢測(cè)方法中,大部分都需要利用反轉(zhuǎn)錄酶將RNA反轉(zhuǎn)錄為c DNA后再進(jìn)行擴(kuò)增檢測(cè)。本論文第三章構(gòu)建了三種無(wú)需加反轉(zhuǎn)錄酶可直接等溫?cái)U(kuò)增RNA的實(shí)驗(yàn)方案,實(shí)現(xiàn)了對(duì)RNA的快速“一步法”檢測(cè)。這幾種方法都利用了本實(shí)驗(yàn)室發(fā)現(xiàn)的Bst DNA聚合酶既具有DNA聚合酶活性又具有內(nèi)在的反轉(zhuǎn)錄酶活性的特點(diǎn);此外,還充分的利用了DNA聚合酶的鏈置換活性及與切刻酶聯(lián)用的擴(kuò)增放大機(jī)理,最終實(shí)現(xiàn)了信號(hào)的多重放大。最優(yōu)方法對(duì)大腸桿菌16S r DNA的檢測(cè)可達(dá)1 amol,對(duì)大腸桿菌16S r RNA在一定的濃度下也可以進(jìn)行有效的檢測(cè)。這些方法均具有在等溫、均相條件下可直接檢測(cè)RNA。在RNA的檢測(cè)中大大縮短反應(yīng)時(shí)間,簡(jiǎn)化操作步驟,降低實(shí)驗(yàn)成本和操作難度。這些新的等溫方法的建立為其他核酸檢測(cè)提供了新方法與新思路,同時(shí)對(duì)即時(shí)檢測(cè)也具有重要意義。
[Abstract]:Nucleic acid as an important genetic information biomolecules, its detection is widely used in food safety, biomedical detection. Polymerase chain reaction (PCR) is one of the most widely used nucleic acid amplification methods. However, PCR technology needs specialized thermal circulator, which limits its application in the field of disease diagnosis and so on. Therefore, it is more sensitive to develop. Rapid isothermal detection of nucleic acid is of great significance. In this paper, a few simple and fast methods of nucleic acid isothermal amplification were constructed, and DNA and RNA were used for rapid detection. Sensitive and specific detection. In Chapter II. In this paper, a new method of isothermal amplification for detection of DNA based on double cut molecular beacon was established. The method was used to detect 16s r DNA of Escherichia coli in real time. When the amount of target is between 100 fmol and 10 amol, the detection limit is 10 amol. By designing the mutation base on the primer for detection, it is proved that the method has strong specificity. In addition, it is a one-pot isothermal chain replacement amplification method, which does not require additional operation steps, greatly simplifies the experimental steps. These advantages enable the method to detect nucleic acid quickly in situ and to detect Point-of-care testing in real time. In current RNA detection methods. Most of them need to use reverse transcriptase to reverse RNA to c DNA before amplification detection. In the third chapter of this paper, three kinds of RNA can be directly isothermal amplified without reverse transcriptase. The fast "one step" detection of RNA is realized. All of these methods make use of the Bst found in our laboratory. DNA polymerase has both DNA polymerase activity and intrinsic reverse transcriptase activity. In addition, the chain replacement activity of DNA polymerase and the amplification and amplification mechanism of DNA polymerase combined with cutting enzyme were also fully utilized. Finally, the multiple amplification of the signal was realized, and the detection of 16s r DNA of E. coli by the optimal method was up to 1 amol. 16s r RNA of Escherichia coli can also be effectively detected at a certain concentration. RNA can be directly detected under homogeneous conditions. In the detection of RNA, the reaction time is greatly shortened and the operation procedure is simplified. The establishment of these new isothermal methods provides new methods and new ideas for other nucleic acid detection, and is also of great significance for real-time detection.
【學(xué)位授予單位】：青島科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：O657.3

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