可攜帶型反轉錄隔絕式恆溫聚合酶連鎖反應檢測鯨豚麻疹病毒感染之研發

Abstract

鯨豚麻疹病毒（CeMV）會引發嚴重的肺炎、腦炎及免疫抑制等症狀，這些症狀嚴重影響鯨豚的游泳及漂浮能力。由於此病毒具有不斷變異的特性，且其宿主範圍日益擴大，未來具備爆發麻疹病毒疫情的可能性，目前太平洋地區由於研究資源不足等原因而缺乏麻疹病毒的相關研究，顯示出開發快速檢測CeMV之方法的重要性。現有的CeMV檢測方法存在多項缺點，包括專業設備需求、仰賴熟練的技術人員等，此外，檢體在運送過程中可能因降解而影響檢測結果。反轉錄隔絕式恆溫聚合酶連鎖反應（RT-iiPCR）可以克服這些問題，其優勢在於檢測快速、裝置便於攜帶，且可用於RNA檢測。由於操作過程相對簡單，即使未經高度訓練的人員也能正確執行，這使得RT-iiPCR成為理想的現場診斷工具。本研究參考已發表的多種CeMV P基因序列（如DMV、BWMV、PWMV和PMV），設計出一組可涵蓋多種病毒株的通用primer與probe，我們使用合成RNA片段來測試RT-iiPCR的靈敏度，並以摻入RNA模板的組織樣本評估其臨床靈敏度。此檢測方法亦在台灣於2024年採集的擱淺鯨豚樣本，以及來自巴西、西班牙及夏威夷的CeMV陽性福馬林固定石蠟包埋（FFPE）樣本中進行驗證。RT-iiPCR的檢測結果與RT-qPCR呈現高度一致，在所有CeMV陽性FFPE樣本中，RT-iiPCR均成功檢測出陽性結果，其Ct值範圍為23至32，顯示RT-iiPCR具有可靠性，且即使是感染程度相對較輕的樣本也能成功檢測。CeMV的監測對於了解流行病學非常重要，並可作為評估整體海洋健康的指標。本研究所開發的CeMV檢測套組不僅能進行快速、現場診斷，更重要的是，在檢測出陽性後我們能得知哪些是具有高度研究與監測價值的陽性樣本，幫助我們有效分配有限資源，如此可以降低後續分析所需的人力、物力與時間成本，提升整體研究效率。因此，此套CeMV RT-iiPCR檢測方法，對於CeMV感染的即時監控以及後續流行病學研究，具有重要意義。

Cetacean morbillivirus (CeMV), a morbillivirus affecting cetaceans, induces a severe combination of pneumonia, encephalitis, and immunosuppression. These symptoms significantly affect the swimming and floating abilities of cetaceans. The evolving nature of the virus and its adaptability to an expanding range of hosts suggest potential morbillivirus epidemics in the future, and there are still knowledge gaps in some areas, especially around the Pacific, emphasizing the need to develop a rapid detection method for CeMVs. Current CeMV detection methods have several disadvantages, including high technical requirements, the need for specialized equipment, and the reliance on skilled personnel. These challenges are compounded by issues such as sample degradation during transportation. Reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) offers a solution to these obstacles. It has advantages such as being rapid, portable and can be used on RNA detection, its relatively simple procedure allows it to be conducted without highly trained technicians. Therefore, RT-iiPCR is an ideal on-site diagnostic tool. In this study, we designed a universal primer and probe set based on published P-gene sequences of CeMV, including DMV, BWMV, PWMV and PMV. Custom synthetic RNA fragments were used to test the analytical sensitivity of the RT-iiPCR, while clinical sensitivity was assessed using tissue samples spiked with RNA templates. The detection method was further validated using CeMV-positive formalin-fixed paraffin-embedded (FFPE) samples, as well as stranded cetacean samples collected around Taiwan in 2024. The performance evaluation of the RT-iiPCR showed substantial agreement with RT-qPCR. CeMV-positive FFPE samples from Brazil, Spain, and Hawaii, were detected CeMV positive in all samples by RT-iiPCR. The Ct values ranged from 23 to 32, indicating that RT-iiPCR is reliable and capable of detecting even mild infections. CeMV surveillance is crucial not only for understanding its epidemiology but also as an indicator of overall ocean health. The CeMV detection kit developed in this study enables rapid and field-deployable diagnosis. More importantly, the initial identification of positive samples allows for the subsequent evaluation of their potential significance in research and surveillance. This capability enhances the strategic allocation of limited resources. This helps reduce the labor, material, and time costs required for subsequent analyses, thereby improving overall research efficiency. Therefore, this CeMV RT-iiPCR assay represents an important tool for the real-time surveillance of CeMV infections and for informing future epidemiological studies.