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

• Main Authors: Tzu-Chia Wang, 王子嘉
• Other Authors: Jing-Tang Yang
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/sk5w8v

碩士 === 國立臺灣大學 === 機械工程學研究所 === 107 === 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.