即時偵測同步定量聚合酶連鎖反應(quantitative real-time polymerase chain reaction, Q-PCR)是利用溫控機構(thermal cycler)將目標雙股去氧核醣核酸(deoxyribonucleic, DNA)進行體外增幅，並以螢光檢測儀(fluorimeter)同時監控增幅過程中DNA嵌合螢光(labeling dye)螢光強度的變化以進行定量分析，用於偵檢及診斷之應用。因此，在本研究提出一微型聚合酶連鎖反應系統，整合一連續式聚合酶連鎖反應晶片，並配合螢光偵測儀進行螢光偵測及定量分析。連續式聚合酶連鎖反應晶片的應用是為了減少在聚合酶連鎖反應過程中過多升降溫時間的浪費，然而整個聚合酶連鎖反應時間仍須取決於試劑的傳輸，因此本研究設計了兩種不同的微流體控制模組用來精準控制試劑傳輸。此外，本研究亦採用一新式型陣列式加熱晶片來改善反應區的溫度均勻性。
該連續式微型晶片包含微流體控制模組及溫控模組，本研究分別利用多重薄膜作動及三組S型氣動式微型幫浦來做為微流體控制模組，用來儲存及快速傳輸試劑通過包含三種不同溫度的反應區，而三不同溫度的反應區則是分別由三個陣列式微加熱器所產生，此三微型加熱器均包含一溫度感測器用以精準地空用反應溫度。此外，可利用微流體控制模組加以調控試劑及樣本於各反應區之反應時間以及所需要的熱循環數目，以最佳化整個聚合酶連鎖反應。而該連續式微型晶片採微流體控制模組與溫控模組分離方式，因此增幅完成後只需更換微流體控制模組即可。本實驗先利用固定式聚合酶連鎖反應晶片進行增生檢測，以確定陣列式微加熱晶片及可拋棄式晶片的可行性。再以連續式微型晶片進行增幅反應，以確定微型晶片能成功增幅目標DNA的特定片段。
最後，本研究採用一螢光偵測儀來進行螢光訊號偵測，本研究利用B型及C型肝炎來驗證微型聚合酶連鎖反應系統的可行性，而B型及C型肝炎的偵測片段長度分別為350及150鹼基對，並利用SYBR Green螢光嵌合進行定量檢測。在增生反應過程中，利用兩種不同的微流體控制模組均能調控試劑於三反應區停留的時間及增生反應所需的循環次數，再以470奈米波長的藍光發光二極體激發螢光嵌合物而發出530奈米波長的綠光訊號。用於檢測增生的樣本濃度由1至105copies/ml，實驗結果顯示利用微型晶片均能成功進行增生反應並偵測螢光訊號。再利用螢光訊號與循環次數關係決定起始循環次數，並計算微型晶片用於B型及C型肝炎增生檢測的增生率分別為1.688及1.742，而增生結果亦利用電泳膠圖加以確認。本研究將微型聚合酶連鎖反應系統用於傳染性疾病的檢測，採用兩種不同微流體控制模組均能以自動化形式完成增生反應，而整合螢光偵測儀則能達到對於型及C型肝炎的定量檢測，因此微型聚合酶連鎖反應系統對於分子診斷能提供一相當有效率的平台。

Quantitative real-time polymerase chain reaction (Q-PCR) systems based on PCR with fluorescence-based detection have been intensively used to amplify and simultaneously quantify a targeted DNA molecule. In this study, a Miniature PCR system integrated with a fluorescence detection system is demonstrated to allow for the detection of infectious diseases. To reduce extra cooling and heating times for a PCR process, two new flow-through PCR microchips have been developed. However, the entire reaction time for a PCR process is still determined by sample transport. To tackle these technical challenges, the present study simplifies the control task by adopting two different microfluidic control modules to precisely drive the sample flow. In addition, a new design of array-type microheaters was adopted to improve the thermal uniformity in the PCR chambers.
The micromachined flow-through PCR chip mainly comprised two micro modules for thermal and microfluidic control. The microfluidic control modules with two different fluid transportation devices by using the multiple membranes and three S-shape micropumps, respectively, were used to rapidly transport the DNA samples through the three heating sections. The microfluidic control modules were also used to adjust the cycle numbers and detention times of the sample in the three temperature control zones, where the PCR thermal cycles were performed. In the micro thermal control module, three individual array-type heating and temperature-sensing sections were integrated to modulate the specific temperature field for three thermal steps of a PCR process. The PCR procedure was also performed by using a stationary PCR chip to confirm the reliability of the thermal uniformity of the array-type microheaters and the feasibility of the disposable micro PCR chip. Then, the flow-through PCR chips with two different microfluidic control modules were also used to perform the PCR procedure.
Finally, a fluorescence detection system was employed for detection of fluorescent signals. The Miniature PCR system was then used for detection of hepatitis B virus (HBV) and hepatitis C virus (HCV) by using SYBR Green fluorescent dye. The PCR sample was excited by a 470 nm blue LED and detected at 530 nm. Biosamples with a concentration ranging from 1 and 105 copies/ml can be successfully detected using this system. By repetitive determinations of known concentrations of the DNA template, the threshold cycles (Ct) was decided, and the amplification efficiency (Eff) was determined. Slab-gel electropherograms also proved that the detection gene for HBV and HCV could be successfully amplified by using the new flow-through PCR chip.
In summary, a Miniature PCR system was developed to allow for the detection of infectious diseases. The new micromachined flow-through PCR chips were designed to perform PCR in an automatic format. With the integration of a fluorescence detection system, quantitative detections of HBV and HCV can be achieved. The development of the Miniature PCR system may provide a useful platform for molecular diagnosis.

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