環保專案成果報告查詢系統

專案計畫編號
經費年度	113
計畫經費	3,230 千元
專案開始日期	2024/03/06
專案結束日期	2024/12/15
專案主持人	賴政佑
主辦單位	國環院檢測技術中心
承辦人	林欣穎
執行單位	益農有限公司
專案分類	環境監測及檢測
中文關鍵字	第三代微生物總基因體定序、重金屬總基因體關聯性分析、分子檢測
英文關鍵字	Third Generation Metagenome Sequencing, Metagenome-wide association study with heavy metal, Molecular diagnostics
協同主持人
共同主持人
計畫聯絡人	余中鼎
計畫聯絡電話	+886 920141249
計畫聯絡信箱	jongding.yu@inong.com.tw

中文摘要	本研究以創新的“先統計，後設計”分子檢測開發策略，透過第三代微生物總基因體分析技術，首先量化土壤樣品中的微生物重金屬抗性基因分布以及微生物代謝途徑分布，透過總基因數據與重金屬濃度數據進行關聯性統計分析(MWAS)，遴選出最佳重金屬代表性生物標誌以開發反應重金屬濃度的引子探針。最後設計、優化及製作q-PCR 篩檢引子及探針並完成土壤確效試驗。統整52 個樣品之重金屬濃度數據及微生物總基因體數據，完成各樣品目標基因量化分析數據及微生物代謝途徑量化分析以進一步進行微生物物種、微生物代謝途徑及目標重金屬抗性基因之關聯性分析，找出與各重金屬有關聯之生物標誌，以抗性基因遴選出6 個具專一性之管制重金屬代表性基因czcC、chrF、pbrA、cznA、cueA 及nccA。針對6 個代表性基因設計、合成及測試引子及探針，最終挑選出鉻目標引子對Cr_chrF_4、鉛目標引子對Pb_pbrA_8 及砷目標引子對As_cueA_4 並製作單一重金屬之q-PCR 引子及探針篩檢套組3 套及複合式重金屬之q-PCR 引子及探針篩檢套組1 套。於先期研究採樣之相同場址採集20 件現場土壤並檢測重金屬，以完成篩檢套組確效試驗，產出目標重金屬鉻、鉛及砷的確效試驗結果。
中文關鍵字	第三代微生物總基因體定序、重金屬總基因體關聯性分析、分子檢測

中文摘要

本研究以創新的“先統計，後設計”分子檢測開發策略，透過第三代微生物總基因體分析技術，首先量化土壤樣品中的微生物重金屬抗性基因分布以及微生物代謝途徑分布，透過總基因數據與重金屬濃度數據進行關聯性統計分析(MWAS)，遴選出最佳重金屬代表性生物標誌以開發反應重金屬濃度的引子探針。最後設計、優化及製作q-PCR 篩檢引子及探針並完成土壤確效試驗。統整52 個樣品之重金屬濃度數據及微生物總基因體數據，完成各樣品目標基因量化分析數據及微生物代謝途徑量化分析以進一步進行微生物物種、微生物代謝途徑及目標重金屬抗性基因之關聯性分析，找出與各重金屬有關聯之生物標誌，以抗性基因遴選出6 個具專一性之管制重金屬代表性基因czcC、chrF、pbrA、cznA、cueA 及nccA。針對6 個代表性基因設計、合成及測試引子及探針，最終挑選出鉻目標引子對Cr_chrF_4、鉛目標引子對Pb_pbrA_8 及砷目標引子對As_cueA_4 並製作單一重金屬之q-PCR 引子及探針篩檢套組3 套及複合式重金屬之q-PCR 引子及探針篩檢套組1 套。於先期研究採樣之相同場址採集20 件現場土壤並檢測重金屬，以完成篩檢套組確效試驗，產出目標重金屬鉻、鉛及砷的確效試驗結果。

中文關鍵字

第三代微生物總基因體定序、重金屬總基因體關聯性分析、分子檢測

類型	檔名	檔案大小	說明	最新版公開日期
期末報告	開發環境綠色化學快篩試劑_成果報告定稿本.pdf	43MB		2025/1/31 上午 12:00:00

摘要	The pioneering“Tally and Design”molecular diagnostics development strategy de-ployed in this research involves first quantitating the distribution of microbial heavy metal resistant genes in contaminated soil samples using Third Generation Metagenome Se-quencing technology and customized bioinformatics pipeline. Next, the Meta-genome-associated analysis (MWAS) runs correlation analysis between the metagenome data and concentrations of eight heavy metals to screen for most representative biomarkers. Lastly, homolog-based primer design will generate sequences to be used to synthesize q-PCR probe, the q-PCR screening primers and probes are designed, optimized, and produced, followed by completion of soil verification experiments. Specifically, the heavy metal concentrations and different dimensions of the metagenomic data of 52 samples were analysis with MWAS. The resultant biomarkers include repre-sentative microbial species and functional genes, namly czcC, chrF, pbrA, cznA, cueA and nccA. Of all the primers and probes designed against these six genes, set Cr_chrF_4, set Pb_pbrA_8 and set As_cueA_4 were selected as the most relevant probe and probe sets and made into three singleplex qPCR kit and one multiplex qPCR kit. Lastly, the kits were validated against 20 new soil samples collected in current study.
英文關鍵字	Third Generation Metagenome Sequencing, Metagenome-wide association study with heavy metal, Molecular diagnostics

摘要

The pioneering“Tally and Design”molecular diagnostics development strategy de-ployed in this research involves first quantitating the distribution of microbial heavy metal resistant genes in contaminated soil samples using Third Generation Metagenome Se-quencing technology and customized bioinformatics pipeline. Next, the Meta-genome-associated analysis (MWAS) runs correlation analysis between the metagenome data and concentrations of eight heavy metals to screen for most representative biomarkers. Lastly, homolog-based primer design will generate sequences to be used to synthesize q-PCR probe, the q-PCR screening primers and probes are designed, optimized, and produced, followed by completion of soil verification experiments. Specifically, the heavy metal concentrations and different dimensions of the metagenomic data of 52 samples were analysis with MWAS. The resultant biomarkers include repre-sentative microbial species and functional genes, namly czcC, chrF, pbrA, cznA, cueA and nccA. Of all the primers and probes designed against these six genes, set Cr_chrF_4, set Pb_pbrA_8 and set As_cueA_4 were selected as the most relevant probe and probe sets and made into three singleplex qPCR kit and one multiplex qPCR kit. Lastly, the kits were validated against 20 new soil samples collected in current study.

英文關鍵字

Third Generation Metagenome Sequencing, Metagenome-wide association study with heavy metal, Molecular diagnostics