本研究提出一整合型微流體晶片系統，可全程自動化地一次進行 4 組反轉錄－聚合連鎖反應，並應用於養殖漁業疾病之快速檢測。此系統包含微型溫控模組與微流體輸送模組，前者藉由微型溫度感測器及微型陣列式自補償加熱器，提供精準、快速且均勻輸出的溫度條件，使生化反應順利進行；後者整合蠕動式微型氣動幫浦與微管道，提供了生物相容性高及可拋棄性，並在程式化控制下傳輸流體，自動銜接兩步驟反轉錄－聚合連鎖反應的各項試劑輸送程序，取代了傳統上繁雜而耗時的人工操作。再者，本研究利用隨機引子，成功地整併反轉錄反應的晶片設計，解決先前研究中試劑浪費問題的同時也一次滿足了多個聚合連鎖反應之需求。也由於反轉錄反應的整併，將反轉錄產物輸送至各個聚合連鎖反應槽的步驟可以同時進行，因此反轉錄反應與聚合連鎖反應槽之間的所有微型氣動幫浦僅需要一個電磁閥同步控制即可。這些微型氣動幫浦的同時作動，加上等長的微管道流阻相等，因此反轉錄產物輸送至聚合連鎖反應槽的同時，也完成了反轉錄產物分配的動作。此一微流體晶片系統是利用微機電系統技術製成（Micro-electro- mechanical-system, MEMS），擁有反應靈敏、檢測速度高、成本低廉、過程自動化、人為失誤降低、樣品與試劑使用量減少等優點。本晶片系統的升降溫速度分別達20℃/ 10℃，優於使用傳統聚合連鎖反應儀器的5℃/ 2℃，且均勻性較傳統區塊式的加熱器為佳。實驗資料顯示，以隨機引子完成反轉錄反應後，確實可在聚合連鎖反應中增幅產物。目前，此系統已針對高經濟價值的石斑魚種，成功地偵測到石斑魚神經壞死症病毒、虹彩病毒與鰻弧菌等 3 種病原的RNA基因片段，以及石斑魚感染病毒時表現量會產生明顯提升的抗病毒Mx蛋白基因（RNA）。以微流體晶片系統對以上RNA片段進行偵測，偵測極限最低皆可達到 101 copies/ml，優於傳統聚合連鎖反應儀器的 103 copies/ml。以微流體晶片系統進行全反應的時間僅需2.5小時，優於使用傳統聚合連鎖反應儀器的4小時。目前使用螢光偵測模組之偵測極限為103 copies/ml，可提供重要的系統可攜性。對於多年來深受病毒型疾病影響而損失慘重的養殖漁業，此一具有可擴充性的晶片系統，足可取代檢測時間冗長、靈敏度等條件亦較差的傳統儀器，為降低經濟損失、遏止病原擴散，以及病原感染機制的相關研究提供更有效率的工具。

This study presents an integrated microfluidic system for fast diagnosis of agricultural diseases with the ability to perform 4 reverse-transcription polymerase chain reaction (RT-PCR) processes at the same time in an automatic format. This system integrates a micro temperature control module and a microfluidic control module. The micro temperature control module comprising micro temperature sensors and array-type microheaters provides precise, fast, and uniform temperature conditions for bio-reaction. The microfluidic control module made of biocompatible materials can transport samples and reagents by using pneumatic micropumps, microvavles and microchannels. Moreover, by using random primers into the reverse-transcription process, the chip design can be simplified and the consumption of reverse-transcription (RT) products in the following multiple polymerase chain reactions (PCR) is also minimized. Since there is only one reverse-transcription reaction involved, the following steps for distributing reverse-transcription products to four PCR chambers can be done simultaneously. When the transportation process between the RT chamber and the PCR chambers was finished, PCR process was then performed to amplify detection genes for each RNA-based virus. This microfluidic chip system fabricated by using micro-electro-mechanical-system (MEMS) technology may have the following advantages, including high sensitivity, fast diagnosis, disposability, low reagent and sample consumption, portability, low power consumption and low cost. The rapid heating/ cooling of this thermocycling system is 20℃/ 10℃. Experimental data showed that the developed system can successfully detect 4 kinds of purified RNA samples, including Nervous Necrosis Virus, Irido virus, Vibrio anguillarum, and grouper Mx protein gene. The detection limit of this system was found to be 101 copies/ ml while the conventional method was 103 copies/ ml. The total reaction time of this developed method was about 2.5 hours. An end-point detection module was also fabricated in this study. The detection limit of the end-point detection module was 103 copies/ ml. The end-point detection module could be integrated into the microcluidic system for its portability. The developed microfluidic system may provide a powerful tool for fast disease diagnosis of RNA-based virus.

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