登革熱是由埃及伊蚊傳播的病毒傳染疾病，主要流行於熱帶和亞熱帶地區。然而，隨著氣候變遷和都市化的影響，目前全球已有超過半數人口處於感染登革熱的風險當中，因此登革熱早期診斷平台的開發對於病患處置及疫情監控至關重要。恆溫式圈環型核酸增幅法（LAMP）是一具有潛力的新興核酸恆溫增幅技術，與傳統的登革熱檢測方法相較之下檢測效能更好且具成本效益，且現行已有若干使用 技術的診斷平台取得政府授權用於病毒感染的檢測。然而，現有的 檢測平台大多依賴於光學方法，因此常發生偽陰性結果，且需昂貴的光學儀器進行即時訊號變化檢測。雖然後續有研究團隊開發場效電晶體（FET）的感測晶片應用於 技術中，但由於臨床端需要使用拋棄式耗材進行檢測，因此未來產線的建立以及高價的單次檢測費用是必須面對的問題。為了避免這些困難，我們提出了一種新的 檢測平台，該平台為使用商品化的印刷 微電極試片，透過測量反轉錄恆溫式圈環型核酸增幅反應後的 差異來進行結果判讀。該試片設計為單次使用，無需校準步驟，量測僅需 分鐘。在測量當中僅需要少量樣本 μl），並且試片可以輕鬆放入常規 管中進行直接 值測量。在本研究中，我們使用登革熱第二型病毒的繼代培養樣本進行了平台的驗證，陽性樣本在61° 進行 反應 分鐘後， 值呈 單位（病毒數量相當於 PFU）的下降，而陰性樣本則僅出現的小於± 單位變化。總結來說，我們提出了一種應用商品化 微電極試片的 檢測平台，該平台有望成為高效且具成本效益的登革病毒檢測技術。其簡單操作的方式和應用彈性使其具有改變核酸檢測方法的潛力，將有助於登革熱的及時診斷和有效控制疫情爆發。

Dengue fever is a viral disease transmitted by Aedes mosquitoes, primarily found in tropical and sub-tropical regions. However, with climate change and urbanization, more than half of the world’s population is at risk of the infection. Therefore, the development of early and reliable diagnostic methods is crucial for patient managements and effective surveillance of the pandemics. Loop-mediated isothermal amplification (LAMP) is a promising nucleic acid amplification technique which is a compelling alternative to conventional diagnostic methods due to their limited effectiveness and affordability. Several LAMP-based diagnostic platforms have been authorized for viral infections. However, most existing LAMP detection platforms rely on optical methods, which is associated with frequent false negatives and the need for expensive real-time monitoring instruments. Some FET-based sensors were introduced in LAMP detection, but the requirement for single use in clinical practices leaves the concerns about establishment of mass production and high cost for each detection. To address these issues, we propose a novel LAMP detection platform that utilizes a commercially available screen-printed pH microelectrode strip. This chip is designed for single-use and waives the need for calibration steps and the pH measurement can be completed within around 1.5 min. It measures the pH difference after the RT-LAMP reaction. Only a small sample volume (<10 μl) is required and can easily fit in regular PCR tubes for direct pH measurement. In this study, the subculture samples of DENV-2 were tested for validation of the presented platform. For positive samples, a pH drops of 0.2-0.5 units (the detectable concentration ranges from 100 ~ 100000 PFU) is observed after the LAMP reaction at 61°C within 30 min, while negative samples show a minimal ±0.1 pH change. Overall, our proposed commercial micro pH electrode strip-based RT-LAMP platform offers a practical solution for efficient and cost-effective dengue virus detection. With its simplicity and scalability, it has the potential to revolutionize nucleic acid detection methods, facilitating timely diagnosis and effective control of dengue outbreaks.

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