- 學位論文
利用重組酶聚合酶擴增結合側流試紙研發可攜式鯨豚產製品檢測套組
Development of a Portable Identification Tool Set Using Recombinase Polymerase Amplification (RPA) with Lateral Flow Strip (LF) for Cetacean Products
摘要
人類對鯨豚的利用已存續非常久，包括作為食用、魚餌以及其他傳統用途。儘管大多國家皆有關於鯨豚利用的法規，非法事件仍經常被揭露，大至鬚鯨小至鼠海豚皆受到影響。然而非法使用鯨豚的規模仍不清楚，使特定物種的存續受到威脅。此外，由於鯨豚製品含有較高的污染累積及人畜共通之病原，食用鯨豚製品對公眾健康存有風險。聚合酶連鎖反應（PCR）搭配定序、快速免疫分析過去皆作為鯨豚肉辨識之工具，但仍受到專業實驗器材、操作人員及樣本新鮮程度需求等限制。 在此研究中，利用重組酶聚合酶擴增（Recombinase Polymerase Amplification, RPA）搭配二合一試紙，研發出一套辨識鯨豚肉及其他組織的可攜式檢驗套組。從鯨豚樣本中粗萃的DNA會由兩對專一的引子擴增，針對粒線體控制區的基因，兩者目標分別為所有鯨豚共有之序列以及鬚鯨獨有之序列。粗萃之DNA在攝氏30至40度間藉由RPA反應十分鐘以擴增，擴增之產物利用試紙可於五分鐘內判讀結果。雙陽性之結果代表受檢驗之產製品源自鬚鯨，而單陽性之結果則代表其源自齒鯨。此套組可正確檢驗四種鬚鯨、十一種齒鯨、海豹、鮪魚和其他六種常見家禽、家畜。與利用蛋白質之免疫分析法相比，此套組亦可檢測經水煮、清炒過的肉品。 藉由此可攜式檢驗套組，不僅能提升可疑產製品檢驗的效率，亦有助於全面的實地調查。此外，檢驗套組也可增進對於非法買賣及消費鯨豚產製品管道的了解，進而對特定鯨豚物種保育及管理政策的建立有所幫助。
並列摘要
Human utilization of cetaceans has persisted for a long time, including food consumption, fishery baits and traditional use. Despite the fact that legislation regarding cetacean use exist in most countries, illegal events were continuously disclosed, affecting numerous species from large whales to small porpoises. Yet the scale of illegal exploitation remained unclear, posing potential threat to the sustainability of certain species. In addition, public health is put at risk regarding the consumption of cetacean-based food products due to high contamination level and zoonotic pathogens. PCR with sequencing and rapid immunoassay were provided as tools for cetacean identification in previous research, but there existed several limitations like demands for laboratory equipment, trained analysts and fresh samples. We developed a portable detection tool set to identify cetacean meat and other tissues based on recombinase polymerase amplification (RPA) with dual lateral flow strip (LF). The DNA crudely extracted from cetacean samples was amplified by two pairs of specific primers based on its mitochondrial control region, with one targeting sequences of all cetacean species and the other targeting only sequences of baleen whales. The DNA was amplified for 10 min at a constant temperature ranging from 30 to 40°C using RPA. Then the amplification products were visualized on the dual LF strip within 5 min. A double positive result indicated that the inspected product belonged to baleen whales while a single positive result implied that belonging to toothed whales. This duplex RPA-LF assay accurately identified four species of baleen whale, 11 species of toothed whales, harbor seal, tuna and six domestic animal species. Compared with protein-based immunoassay, this duplex RPA-LF assay was also effective when using boiled and pan-fried samples. With this portable and accessible detection tool set, inspecting suspicious cetacean products may become more efficient and facilitate the implementation of an extensive field survey. Moreover, it can improve the understanding of the pathway of illegal trade and consumption of cetacean products. A more comprehensive insight of the cetacean consumption is essential for establishing feasible management policy toward certain vulnerable species.