蘭花病毒之核酸萃取、擴增及偵測整合於紙流道之病害診斷

Integrated Nucleic Acid Extraction, Amplification, and Detection on Paperfluidics for Diagnostics of Orchid Virus Disease

王子嘉(Tzu-Chia Wang)

指導教授 ： 楊鏡堂

共同指導教授 ： 廖英志(Ying-Chih Liao)

國立臺灣大學/工學院/機械工程學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201901877

摘要

本研究結合了的核酸萃取、擴增與偵測並應用於蘭花病毒的分子診斷 (molecular diagnostics) 中，除了將診斷技術整合於紙流道 (paperfluidics) 上，更落實了紙基底流體系統中低成本、易操作、方便攜帶的特色，同時，縮短了傳統分子診斷技術中繁雜的步驟與冗長的操作時間，也擺脫了精密儀器的需求。本研究針對蘭花主要的兩種病毒進行診斷，達到即時的定點照護 (point of care)，實驗中透過已知的技術與方法研發出創新的應用。
本研究從紙流道材質中找出纖維素 (cellulose) 對於核酸有集中且濃縮的特性，並發現漸擴漸縮型的橢圓形流道可以純化出最多的核酸量，整個萃取過程僅需要10分鐘，比傳統管柱萃取法的時間快上12倍，同時擺脫了繁複設備的需求；純化後，以目標的病毒基因進行擴增 (amplification)，本研究以TwistDx開發的恆溫擴增技術－重組酶聚合酶擴增 (RPA) 與本實驗室中設計的引子 (primer) 進行擴增，整體擴增的時間僅剩30分鐘，且溫度只需要39°C就可以有效地執行，若利用傳統的分子擴增 (RT-PCR) 需耗費160分鐘的時間，且需要溫度控制95°C-55°C-72°C循環，除了以蘭花的肌動蛋白 (Actin) 作為實驗的控制組，更針對蕙蘭嵌紋病毒 (CymMV) 及齒舌蘭輪斑病毒 (ORSV) 兩大病毒的基因片段進行擴增；最後偵測 (detection) 方面，以側層流裝置 (lateral flow strip) 進行分析，結合生物標記物Biotin與FAM，同時跑電泳 (gel electrophoresis) 驗證其結果。整體而言，萃取配方以胍硫氰酸鹽 (guanidine thiocyanate) 的呈現效果最佳，擴增方法以RT-RPA可以強烈擴增出蘭花RNA病毒的訊號，偵測以側層流條即可達到簡單又快速的診斷。
本文探討核酸於紙材質的流動特性與分子診斷中各階段的方法分析，最後將其整合於紙平台中，製作出簡單操作且方便攜帶的紙基底元件，期望未來不僅能操作於實驗室中，更能於田野中提供農業即時診斷。

關鍵字

分子診斷 ； 紙流道 ； 定點照護 ； 重組酶聚合酶擴增 ； 側層流裝置

並列摘要

In the study, we combined the extraction, amplification, and detection of nucleic acids in molecular diagnostics, and integrated them into paper-based microfluidics (paperfluidics) which was low-cost, lightweight, and portable. Paperfluidics reduced the complicated process and operation time compared to traditional molecular diagnostic technology and did not require precise instruments. This study developed point of care (POC) device that combined known technology and innovational methods, which could diagnose two major orchid viruses.
First, the results showed that elliptic paperfluidics made by cellulose-based paper could capture and concentrate nucleic acids rapidly. The extraction process took only 10 minutes which was 12 times faster than using normal column purification. The second step was to amplify targeted virus genes by using recombinase polymerase amplification (RPA) at a constant temperature of 39°C. Compared with conventional amplification, polymerase chain reaction, RPA merely takes 30 minutes to amplify. Finally, using commercially available lateral flow strips for detection, we demonstrated the ability of the device to detect the two viruses CymMV and ORSV. Meanwhile, the results of the amplification reaction were analyzed by agarose gel electrophoresis. The best buffer was adding guanidine thiocyanate to extraction. Moreover, isothermal amplification and lateral flow assay integrated on the paper-based microfluidics achieved simple and rapid molecular diagnostics.
The study successfully transformed molecular diagnostics into a portable paperfluidic device. We hope that the paper-based device will be used not only in the laboratory but also in the environments with limited resources such as farmland.

並列關鍵字

molecular diagnostics ； paperfluidics ； point of care ； recombinase polymerase amplification ； lateral flow strip

參考文獻

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