本文關鍵詞： 等溫擴增 信號放大 分子信標 核酸檢測 POCT　出處：《青島科技大學》2016年碩士論文　論文類型：學位論文

【摘要】：核酸作為重要的遺傳信息生物分子,其檢測被廣泛的應用在食品安全、生物醫(yī)學檢測、環(huán)境檢測等諸多領域。聚合酶鏈式反應(Polymerase Chain Reaction,PCR)是目前應用最廣泛的核酸擴增檢測方法之一,然而PCR技術需要專門的熱循環(huán)儀,限制了其在疾病的即時診斷等領域的應用。因此,發(fā)展更靈敏、快速的核酸等溫檢測方法具有重要意義。本論文構建了幾種簡單、快速的核酸等溫擴增檢測新方法,分別對DNA和RNA進行了快速、靈敏和特異地檢測。在第二章中,本論文建立了一種基于雙切口分子信標的等溫擴增檢測DNA新方法的研究。用該方法對提取的大腸桿菌16S r DNA進行實時熒光檢測,當檢測靶標的量在100 fmol到10 amol之間時呈良好的線性,最低檢測限為10 amol;通過在引物上設計突變堿基進行檢測,驗證了該方法具有較強的特異性;并用該方法在復雜體系中檢測時呈現(xiàn)較強的抗干擾能力。此外,它是一鍋式的等溫鏈置換擴增方法,不需要額外的操作步驟,極大的簡化了實驗步驟,并降低了樣品污染的可能性。它的這些優(yōu)點使該方法在核酸的現(xiàn)場快速檢測及即時檢測(Point-of-care testing,POCT)中更具有實用性。目前的RNA檢測方法中,大部分都需要利用反轉錄酶將RNA反轉錄為c DNA后再進行擴增檢測。本論文第三章構建了三種無需加反轉錄酶可直接等溫擴增RNA的實驗方案,實現(xiàn)了對RNA的快速“一步法”檢測。這幾種方法都利用了本實驗室發(fā)現(xiàn)的Bst DNA聚合酶既具有DNA聚合酶活性又具有內在的反轉錄酶活性的特點;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)用的擴增放大機理,最終實現(xiàn)了信號的多重放大。最優(yōu)方法對大腸桿菌16S r DNA的檢測可達1 amol,對大腸桿菌16S r RNA在一定的濃度下也可以進行有效的檢測。這些方法均具有在等溫、均相條件下可直接檢測RNA。在RNA的檢測中大大縮短反應時間,簡化操作步驟,降低實驗成本和操作難度。這些新的等溫方法的建立為其他核酸檢測提供了新方法與新思路,同時對即時檢測也具有重要意義。
[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.
【學位授予單位】：青島科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：O657.3

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