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年度	103	專案性質	實驗性質
專案類別	研究專案	研究主題	調查
申請機構	國立屏東科技大學	申請系所	環境工程與科學系
專案主持人	趙浩然	職等/職稱	教授
專案中文名稱	應用雙色冷光提升戴奧辛生物快速篩選技術於土壤污染模場試驗先行評估計畫
中文關鍵字	報導基因法；戴奧辛；雙色冷光
專案英文名稱	Using dual luciferase to increase the stability and sensitivity for screening dioxin samples in Ad-DR bioassay
英文關鍵字	reporter gene assay；dioxin；dual luciferase
執行金額
執行期間	2014/4/25 至 2014/11/24
計畫中文摘要	本計畫以多方合作的方式進行土壤污染模場試驗前的評估，在本團隊開 發之 Ad-(6X)DR bioassay (DRE-H4IIE 細胞)的基礎上，由國家衛生研究院開 發雙色冷光 Ad-(dual)DR bioassay，屏東科技大學負責雙色冷光 Ad-(dual)DR bioassay 之生物快篩檢測戴奧辛標準品與環境樣品，以及後續的修正與改進 工作，藉此增進雙色冷光 DRE-(dual)H4IIE 細胞的穩定性與敏感度，以開發 快速、有效而低成本的本土戴奧辛生物篩檢技術。目前的研究成果顯示， Ad-(dual)DR bioassay病毒的multiplicity of infection (MOI)為2時為本研究最佳 實驗條件，2,3,7,8-TCDD 檢量線最佳回推區間為 4 ~ 120 pM (RSD = 4 - 22%)，EC50 為 54.3 至 55.8 pM。Ad-(dual)DR bioassay 測試 25 件次環境基質 標準品(Certified Reference Materials, CRM)，其測值與 CRM 濃度(466 pg-WHO1998-TEQ/g d.w.)值相比時高了 1.25-1.61 倍。19 件次真實土壤與底泥 樣本測試與 HRGC/HRMS 平均比值為 2.59 倍，兩者具有良好之關聯性 (R2 =0.957, p<0.001)且無偽陽性之問題。40 件次樣本與 XDS-CALUX 比對平 均比值為 0.974 倍，參照國際文獻 XDS-CALUX 與 HRGC/HRMS 平均比值 約為 1.5-2.6 倍，顯示本研究樣本回推濃度與 HRGC/HRMS 相似。本研究所 開發之 Ad-(dual)DR bioassay 病毒使用量低，在中、高濃度樣本穩定度與回 推能力之表現良好，雖無法完全改善低濃度樣本穩定度表現，但比照現行規 範濃度 1000 ng TEQ/kg d.w. 是非常適合推廣與應用的。
計畫英文摘要	This project is a multi-collaborated work to evaluate soil dioxins contamination before the pilot study. According to the basis of previously establishing Ad-(6X)DR bioassay (DRE-H4IIE cells) by our research team, the National Health Research Institutes develops dual luciferase detection system for detecting dioxin like compounds, namely Ad-(dual)DR bioassay, and the team members in National Pingtung University of Science and Technology test the dual system for application in detection of soil dioxins contamination and modify this system for the future dioxins survey studies. We hope our collaboration elevate the stability and sensitivity of dioxins measurements in Ad-(dual)DR bioassay to accesses a fast-screen, efficient, low-cost, and local biotechnique for AhR reporter gene assay. The current result show that the optimum multiplicity of infection (MOI) for Ad-(dual)DR bioassay is 2. The best period of the 2,3,7,8-TCDD standard curve for the Ad-(dual)DR bioassay is between 4 and 120 pM (relative standard deviation (RSD) =4-22%. The values of EC50 in Ad-(dual)DR bioassay with MOI of 2 are ranged from 54.3 to 55.8 pM. Twenty five Certified Reference Materials (CRM) samples are examined by Ad-(dual)DR bioassay and their measurements have 1.25-1.61 folds higher than those are measured by HRGC/HRMS (466 pg-WHO1998-TEQ/g d.w.). The mean value measured by Ad-(dual)DR bioassay was 2.59 times higher than that by HRGC/HRMS in soil samples. The good correlation (R2 = 0.957, p<0.001) was found between Ad-(dual)DR bioassay and HRGC/HRMS without false negative results. Our ratio of Ad-(dual)DR bioassay v.s. HRGC/HRMS was similar to those of XDS-CALUX v.s. HRGC/HRMS in the previous studies (Ratio=1.5-2.6). Although the small amounts of the recombinant adenovirus were used in the present study, the performance of the TCDD standard curves was robust and stable in the higher dioxin contaminated soil samples. The stability of low dioxin concentrations in soil samples was varied. It is suitable for application and development of Ad-(dual)DR bioassay in soil surveillance based on the current dioxins standard (1000 ng TEQ/kg d.w.) for the dioxin contaminated soils