利用螢光共振能量轉換與恆溫RNA擴增之快速即時核酸偵測系統

Abstract

本研究發展出一種具備快速即時性、高準確性、高靈敏度之恆溫特定RNA序列偵測，更對此方法建構出造價低廉的核酸序列檢測儀器。 在本研究中指出使用恆溫RNA 擴增進行特定RNA 基因序列擴增，在90 分鐘內利用1 ng total RNA 將特定基因（TRIM28）放大至 108 倍以上，證實實驗具有相當高的靈敏度。進而將恆溫RNA 擴增與螢光能量共振轉換（Fluorescence Resonance Energy Transfer，FRET）方法做結合，進行即時性的螢光訊號偵測，藉此判斷特定RNA 序列的存在。為求提高增加實驗之準確性，特別將探針增加一組，個別標記有FRET 效應之不同螢光分子，必須兩個探針皆正確的鍵結在序特定列上，才視為真正偵測到特定基因序列，藉此增加其正確鍵結的困難度，以求得即時性、高準確性、高靈敏度之恆溫特定RNA 序列偵測。 實驗結果顯示，本論文成功地結合恆溫RNA 擴增與FRET，開發出具備即時性、高準確性、高靈敏性之核酸序列檢測方法。 由於市售之量子點顆粒過大使得FRET 並沒有顯著表現。並且進一步進行量子點的表面改質，其結果穩定性不夠，也無法應用於本研究中。 因此本論文所設計之激發光源與儀器，寄望未來量子點的顆粒能縮小，且具備更穩定的型態，使得本研究發展之具備即時性、高準確性、高靈敏度之即時性恆溫特定RNA 序列偵測，能夠在靈敏度達到更進一步。

In this thesis, we develop a detecting method with high accuracy and high sensitivity for specific RNA sequences in isothermal environment. We construct a low-cost and real-time system for detection specific RNA sequences by using this method. Isothermal RNA amplification is a powerful tool to amplify specific sequences of RNA. It can successfully amplify as low as 1 ng of total RNA in 90 minutes. Therefore, we integrate isothermal RNA amplification and Fluorescence Resonance Energy Transfer (FRET) methods to real-time detect fluorescent signals for specific RNA sequences. For increase specificity, two hybridization probes which can bind to the same gene are used in our experiments to increase the accuracy. Base on this study, a novel method with higher accuracy and sensitivity for detecting specific RNA sequence in isothermal environment can be achieved. The results shown that the FRET signal of TRIM28 and UCHL1 genes have been detected in 40-60 min. Therefore, we have successfully setup a new low-cost, real-time detection system, based on combined Isothermal RNA Amplification and FRET.