- 學位論文
河魨魚種之基因探針即時定量法開發暨TTX基因毒理研究之探討
Studies on quantitative gene probe development for pufferfish and genotoxicity of TTX
摘要
臺灣產魨科魚種約30餘種，大部分含有致命性毒素河魨毒 (Tetrodotoxin; TTX)，一旦於加工製造過程中被誤用或攙雜未經許可之魚肉中，若含帶有不等比例的有毒魨科魚肉，就構成商業詐欺行為和食品安全之公共問題，為防止這些問題的發生，有必要盡速建立快速且準確的定性定量檢測方法，如此有利於生產過程中即時確認加工食品之原料魚種以確保國人食用安全性。 本論文首先針對 Lagocephalus 屬和 Takifugu 屬的河魨，進行 cytochrome b (cyt b) 和 cytochrome c oxidase I (COI) 基因序列之分析比較，結果顯示 L. 屬在 cyt b 及 T. 屬在 COI 基因區塊具有屬特異性和專一性，進一步設計屬專一性引子對分別為: L. 屬: LF-cytb74163/LR-cytb86890、T. 屬: TF-COI31332/TR-COI41233；屬特異性探針分別為: L. 屬: L-probecytb、T. 屬: T-probeCOI，其增幅片段在 L. 屬為 150 bp 及在 T. 屬為 121 bp，並進行引子對及探針專一性及靈敏度之試驗，由結果顯示引子對及探針均無與其他魚種有交互反應，而 DNA 濃度之靈敏度極限為 L. 屬和 T. 屬：10-2 ng/μL，故此兩組引子對及探針非常適合作為兩屬魨科魚種之鑑別應用。 利用上述之檢測條件針對實際收集的市售調味水產加工製品進行檢測，並成功鑑別出早期收集之香魚片樣品皆為魨科魚種製成，而近 2 年來實際收集臺灣北中南東部等地之樣品則均未檢測出含有魨科魚種，結果顯示此即時定量檢測條件可適用於鑑別魨科魚種製成的加工品，並可作為準確的定量定性分析技術。 而一旦河魨毒被攝食進入人體後，其中毒機制主要為抑制末梢神經之鈉腔體的神經訊息傳導。假使當攝入之河魨毒劑量非常微量時，是否與單一核苷酸產生交互反應，進一步生成 DNA adduct 而導致肝細胞毒性的作用，值得深入探討。故此河魨毒之致毒機制具有突破性之學術研究價值。 本研究以體外試驗 (in vitro) 方式進行河魨毒與單一核苷酸 dGMP 的交互反應並以 HPLC 進行檢測分析後，結果顯示河魨毒並不與 dGMP 產生反應作用，本部分實驗的數據未來可作為河魨毒基因毒理作用之基礎背景資料。 綜合上述，本篇論文成功開發魨科魚種之即時定量法基因探針法，未來可應用在鑑別加工食品之原料物種上，尤其是攙雜魨科魚肉原料之樣品，可提供作為重要之定量分析技術。另外，亦提供河魨毒基因毒理之基礎性資料，可供未來其他相關研究之用，此部分研究亦是首例，故為突破性學術探討及具實用價值之研究。
並列摘要
The majority 30 species of Taiwan pufferfish species are born with tetrodotoxin (TTX) and non-economic value due to small quantity of production. Due to fraud or incorrect manufacturing processes, different proportions of unexpected or pufferfish muscle may be incorporated. The volatility of these factors may lead to counterfeit and concerned issue for public food safety. To solve these problems, a rapid qualitative and quantitative method which can accurately and quantitatively authenticate the source fish species under different of production processing should be established as soon as possible. The first study was focused on the gene map analysis of cytochrome b (cyt b) and cytochrome c oxidase I (COI) for Lagocephalus genus and Takifugu genus. The results show the strong genus-specificity and sensitivity on cyt b gene of L. genus and COI gene of T. genus. The further designed genus specific primers and genus Taqman probes are LF-cytb74163 / LR-cytb86890 and T. genus: TF-COI31332 / TR-COI41233, L- probe cytb and T- probe COI of L. genus and T. genus respectively. The amplified length of fragments was 150 base pairs for L. genus and 121 base pairs for T. genus. Specificity and sensitivity of specific primers and genus Taqman probes were also tested. These results demonstrated the primers and Taqman probes were able to discriminate efficiently of L. and T. genus individually and did not have reaction with other species. The detection limit of DNA concentration is 10-2 ng/μL of L. genus and T. genus. Furthermore, apply the above method to identify the commercially products is successfully identify the dry-dressed fish fillets collected before which were all made from pufferfish. The recent collection of these two years from Taiwan’s northern, central, southern and eastern the samples were all not made from pufferfish. It was shown the primers and Taqman probes were able to identificate the processed products and coupled with Real-time PCR is an accurate qualitative analysis technique. Once the TTX was ingested into the body, the toxicity mechanism is blocking the sodium channels entry of sodium ion on neuronal membranes. Suppose low dose-TTX has been ingested that forms a number of different mononucleotide and dinucleotide adducts in DNA, TTX-derived DNA adducts may cause liver cell toxicity. This study actually provided a very important reference on TTX toxicogenomics analysis. In our study, the in vitro test of TTX interacted with a single nucleotide dGMP via the HPLC analysis was done. It was shown that TTX did not have reaction with dGMP. Our data can be used as the basic information of TTX toxicogenomics. To summarize our study actually provided a very important reference on quantitative analysis of sensitive and reliable analyzing method for food safety and especially for adulterated pufferfish products. In addition, the TTX of genotoxicity also provide the basis toxicogenomics information for future related research purposes, and this is also the first study. Our study actually proved groundbreaking and practicability on science research.