日本鰻快速鑑別檢驗方法之建立   全文下載

Rapid Identification of Japanese Eel (Anguilla japonica) in Food Using Real-time PCR

日本鰻（Anguilla japonica）在臺灣被廣泛養殖，惟養殖所需鰻苗仰賴天然撈捕，近年來野生日本鰻資源數量銳減，導致成鰻銷售價格上漲，而同為鰻鱺屬（Anguilla spp.）之鱸鰻（Anguilla marmorata）及太平洋雙色鰻鱺（Anguilla bicolor pacifica）亦在臺灣被大量養殖，另外除鰻鱺屬鰻魚外亦有繁星糯鰻（Conger myriaster）等，皆可用於製作鰻魚料理。不同鰻魚物種經加工後外觀相似難以分辨，鰻魚雖能以生物DNA條碼定序方法進行物種鑑別，但若檢體經高度加工或混加不同物種則不易檢驗，本研究針對日本鰻建立即時聚合酶連鎖反應（real-time PCR）專一性檢驗方法，利用粒線體全基因序列進行比對分析，選定cytochrome b（Cyt b）設計專一性引子及探針，以蒐集之135個魚種DNA進行real-time PCR專一性測試，結果僅目標物種產生專一性陽性增幅反應；經測試最低檢測濃度可達0.001%（1.5 pg DNA），為確認本方法適用性，以17件市售鰻魚檢體進行測試，有15件檢體結果為日本鰻呈陽性反應，而2件陰性反應檢體分別為美洲鰻鱺（Anguilla rostrata）及繁星糯鰻，本方法可應用於日本鰻之快速檢驗，把關國人消費權益。

Japanese eel (Anguilla japonica) is widely cultured in Taiwan, while the supply of eel fry relies on capture from the wild. In recent years, the population of Japanese eel has declined, and it caused the increase in the price of mature eel. Other Anguilla ssp. species such as marbled eel (Anguilla marmorata) and Indian shortfin eel (Anguilla bicolor), also have potential for extensive aquaculture in Taiwan. While Conger eel (Conger myriaster) is also used to make eel dishes. Processing results in increased difficulties of species identification and may lead to mislabeling and substitutions of species. DNA barcoding technique has been used for eel species identification. However, it becomes challenging when samples are highly processed or contain mixed species. This study developed a Japanese eel specific real-time PCR method. The primers and probes were designed specifically to the gene encoding cytochrome b (Cyt b). Specificity was tested and confirmed by using 135 fish species commonly found in the markets of Taiwan. Only the target species showed positive amplification signal. The detection limit was as low as 0.001% at 1.5 pg of DNA. We collected 17 commercial eel samples and tested accordingly. 15 samples tested positive for Japanese eel, and two negative samples were confirmed as American eel (Anguilla rostrata) and Conger eel (Conger myriaster). This method provided a rapid, sensitive and reliable detection tool for the identification of Japanese eel products, allowing the detection of fraudulent and unintentional mislabeling of these species.