結合微光譜儀之可攜裝置於恆溫式圈環型核酸增幅的檢測

A Portable device integrated with a microspectrometer for the detection of loop-mediated isothermal amplification

潘俊祐(Pan Chun-Yu)

指導教授 ： 陳志堅

國立屏東科技大學/工學院/生物機電工程系所/碩士(2019年)

https://doi.org/10.6346/NPUST202000096

摘要

環境的變化造成許多突變的病毒出現，因此及早發現病源作出防疫對策是相當重要的，在檢疫上大多使用核酸複製法進行檢測，又以聚合酶連鎖反應(polymerase chain reaction，PCR)與恆溫式圈環型核酸增幅法(loop-mediated isothermal amplification，LAMP)最為常見，LAMP是以恆溫60°C~65°C的方式加熱15分至1小時不等，反應物經過LAMP反應過後會產生白色沉澱物，可以使用肉眼觀察到白色沉澱物以判斷擴增成功，但礙於每個人的肉眼看到有所不同，可能導致判斷失誤，因此本研究選擇光譜儀代替人眼進行檢測，LAMP除了反應過後會產生白色沉澱物進行觀察外，亦可使用螢光染劑進行染色，若成功增幅去氧核醣核酸(deoxyribonucleic acid，DNA)時，反應溶液會在激發光下發出螢光，可幫助判斷反應結果，對於光譜儀的偵測也更加容易，本研究嘗試使用自製加熱裝置進行持續的恆溫供應，以供LAMP所需的溫度並持續一個小時加熱，並將整個過程的溫度數據上傳至雲端資料庫進行記錄，以便記錄與機器調整參考，檢測方法則搭配光譜儀進行即時的擴增檢測，省去使用凝膠電泳進行檢測的步驟，以達到擴增與即時檢測的目的，本研究先以凝膠電泳驗證自製加熱機台的可行性，使用SYBR Green I Nucleic Acid Stain螢光染劑將樣本進行染色後在暗室中進行LAMP並使用波長 365 nm 之紫外光照射樣本管，過程中以光譜儀代替人眼進行螢光檢測，變色過程中紀錄光譜訊號並分析，成功檢測出螢光的樣本再做凝膠電泳驗證是否成功擴增。

關鍵字

恆溫式圈環型核酸增幅法 ； 螢光檢測 ； 光譜儀 ； 雲端資料庫

並列摘要

Changes in the environment have caused the emergence of many mutated viruses. Therefore, it is very important to find the source of the disease and take preventive measures. In quarantine, nucleic acid replication is mostly used for detection, and polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) are the most common. LAMP is heated at a constant temperature of 60 ° C ~ 65 ° C for 15 minutes to 1 hour. After reacting with LAMP, a white precipitate will be generated. The white precipitate can be observed with the naked eye to judge the successful to amplification. But because each person's naked eye sees it differently, it may lead to misjudgment. In this study, a spectrometer was selected instead of human eyes for fluorescence observation. In addition to the observation of white precipitates after the reaction, LAMP can also be stained with fluorescent dyes. If deoxyribonucleic acid (DNA) is successfully amplified, the reaction solution will emit fluorescence under excitation light. The reaction solution emits fluorescence under the excitation light, which can help judge the reaction result, and it is easier to detect for the spectrometer. This study attempts to use a self-made heating device for continuous constant temperature supply to the temperature required by LAMP and heating for one hour. And upload the temperature data of the whole process to the cloud for recording, For record and machine adjustment reference. Detection method is real-time amplification detection for spectrometer .No need to use gel electrophoresis to get the detection results, to achieve the purpose of real-time detection. In this study, the feasibility of a self-made heating machine was first verified by gel electrophoresis. Samples were stained with SYBR Green I Nucleic Acid Stain fluorescent dye, LAMP was performed in a dark room, and the sample tube was illuminated with invisible ultraviolet light at a wavelength of 365 nm. In the process, a spectrometer is used instead of the human eye for fluorescence detection. The spectral signal is recorded and analyzed during the color change process. Using gel electrophoresis comparison of samples that successfully detected fluorescence.

並列關鍵字

Loop-mediated isothermal amplification ； fluorescent detection ； spectrometer ； cloud database stores data

參考文獻

[1] Mullis, K. B., Erlich, H. A., Arnheim, N., Horn, G. T., Saiki, R. K., & Scharf, S. J. (1987). U.S. Patent No. 4,683,195. Washington, DC: U.S. Patent and Trademark Office.

[2]Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., &Hase, T. (2000).Loop-mediated isothermal amplification of DNA.Nucleic Acids Research, 28(12), e63-e63.

[3]Liu, C., Geva, E., Mauk, M., Qiu, X., Abrams, W. R., Malamud, D., Curtis, K., Owen, S. M., &Bau, H. H. (2011).An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases.Analyst, 136(10), 2069-2076.

[4]Wang, C. H., Lien, K. Y., Wang, T. Y., Chen, T. Y., & Lee, G. B. (2011). An integrated microfluidic loop-mediated-isothermal-amplification system for rapid sample pre-treatment and detection of viruses. Biosensors and Bioelectronics, 26(5), 2045-2052.

[5] Wang, Y., Kang, Z., Gao, Y., Qin, L., Chen, L., Wang, Q., Li, J., Gao, H., Qi, X., Lin, H.,& Wang, X. (2011). Development of loop-mediated isothermal amplification for rapid detection of avian leukosis virus subgroup A. Journal of Virological Methods, 173(1), 31-36.