環保專案成果報告查詢系統

專案計畫編號	NERA154112001
經費年度	112
計畫經費	3,850 千元
預算科目	提升土壤及地下水污染調查及整治技術工作
專案開始日期	2023/05/23
專案結束日期	2023/11/15
專案主持人	賴政佑
主辦單位	國環院檢測技術中心
承辦人	林哲雄
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、重金屬抗性基因分布、分子檢測
英文關鍵字	Third Generation Metagenome Sequencing, Heavy metal resistance gene profile, 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	04-22331742#
計畫聯絡信箱	jongding.yu@inong.com.tw

中文摘要	本研究以“先統計，後設計”的ｑ－ＰＣＲ探針開發策略，透過第三代長片段微生物總基因體定序及資訊分析技術，量化土壤樣本中的微生物重金屬抗性基因分布，搭配樣本的管制重金屬濃度進行關聯性統計分析，遴選出能反映重金屬濃度的最佳重金屬代表性基因，並以代表性基因設計同源引子，製作適用於篩檢土壤重金屬ｑ－ＰＣＲ探針試劑。本研究首先針對重金屬汙染管制場址(台南及彰化)的土壤樣品，以及解除列管場址(台南)及非列管場址(南投)的土壤樣品進行重金屬檢驗以及微生物總基因體分析。另外，本研究亦納入先前「微生物菌相、訊號基因與訊號因子檢測鑑定分析」研究案樣品土壤重金屬檢測及微生物族群總基因體分析資料，進行微生物重金屬抗性基因量化分析。基因量化分析數據與重金屬濃度數據的相關性分析結果遴選出鉻ｃｈｒＦ、銅ｃｏｐＧ、鎳ｎｉｋＣ、鉛ｐｂｒＲ及鋅ｚｉｔＢ／ｙｂｇＲ等5項重金屬的代表性抗性基因，再進一步設計共25組同源引子及製作ｑ－ＰＣＲ探針，ｑ－ＰＣＲ檢測結果顯示22組ｑ－ＰＣＲ探針的檢測Ｃｔ值與重金屬濃度正相關，最後將其中4組ｑ－ＰＣＲ探針製作成試劑。本次研究驗證了藉由微生物總基因體數據分析可大幅縮短微生物ｑ－ＰＣＲ檢測土壤汙染物的探針開發時間。未來，微生物ｑ－ＰＣＲ篩檢土壤重金屬方法可持續擴大統計分析採用的樣品數量以提高對應汙染物檢測濃度的精準度，以利應用至現場測試。
中文關鍵字	第三代微生物總基因體定序、重金屬抗性基因分布、分子檢測

中文摘要

本研究以“先統計，後設計”的ｑ－ＰＣＲ探針開發策略，透過第三代長片段微生物總基因體定序及資訊分析技術，量化土壤樣本中的微生物重金屬抗性基因分布，搭配樣本的管制重金屬濃度進行關聯性統計分析，遴選出能反映重金屬濃度的最佳重金屬代表性基因，並以代表性基因設計同源引子，製作適用於篩檢土壤重金屬ｑ－ＰＣＲ探針試劑。本研究首先針對重金屬汙染管制場址(台南及彰化)的土壤樣品，以及解除列管場址(台南)及非列管場址(南投)的土壤樣品進行重金屬檢驗以及微生物總基因體分析。另外，本研究亦納入先前「微生物菌相、訊號基因與訊號因子檢測鑑定分析」研究案樣品土壤重金屬檢測及微生物族群總基因體分析資料，進行微生物重金屬抗性基因量化分析。基因量化分析數據與重金屬濃度數據的相關性分析結果遴選出鉻ｃｈｒＦ、銅ｃｏｐＧ、鎳ｎｉｋＣ、鉛ｐｂｒＲ及鋅ｚｉｔＢ／ｙｂｇＲ等5項重金屬的代表性抗性基因，再進一步設計共25組同源引子及製作ｑ－ＰＣＲ探針，ｑ－ＰＣＲ檢測結果顯示22組ｑ－ＰＣＲ探針的檢測Ｃｔ值與重金屬濃度正相關，最後將其中4組ｑ－ＰＣＲ探針製作成試劑。本次研究驗證了藉由微生物總基因體數據分析可大幅縮短微生物ｑ－ＰＣＲ檢測土壤汙染物的探針開發時間。未來，微生物ｑ－ＰＣＲ篩檢土壤重金屬方法可持續擴大統計分析採用的樣品數量以提高對應汙染物檢測濃度的精準度，以利應用至現場測試。

中文關鍵字

第三代微生物總基因體定序、重金屬抗性基因分布、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	微生物q-PCR篩檢土壤重金屬技術開發_成果報告定稿本_含大綱及附錄.pdf	16MB		2023/12/1 上午 12:00:00

摘要	In this study, we adopted the “Tally and Design” strategy to develop q-PCR probes which can serve as an indicator of heavy metal concentrations in the soil. The strategy first involves extracting gene frequencies of candidate microbial heavy metal resistant genes from Third Generation long read metagenome analysis of the soil samples. Subsequently, heavy metal resistant genes that demonstrate strong correlation with concentration of heavy metal are selected as representative genes of respective heavy metal. Homolog q-PCR primers and probes are then designed against these representative genes and validated. As results shown, correlation analysis between heavy metal concentrations and gene frequencies of soil samples from heavy metal contaminated areas (Tainan City and Changhua) City, and soil samples from non contaminated areas (Tainan City and Nantou City) identified chrF (Chromiun), copG (Copper), nikC (Nickel), pbrR (Lead) and zitB/ybgR (Zinc) as the representative genes. Subsequently, among the 25 sets of qPCR probe were designed and validated, 22 sets demonstrated Ct values that are positively correlated to the concentration of heavy metals. In conclusion, this study has successfully shown that Third Generation long read metagenome analysis can greatly shorten the development process of qPCR probes. Moving forward, the accuracy of the probes can be further improved by including more samples in the correlation analysis.
英文關鍵字	Third Generation Metagenome Sequencing, Heavy metal resistance gene profile, Molecular diagnostics

摘要

In this study, we adopted the “Tally and Design” strategy to develop q-PCR probes which can serve as an indicator of heavy metal concentrations in the soil. The strategy first involves extracting gene frequencies of candidate microbial heavy metal resistant genes from Third Generation long read metagenome analysis of the soil samples. Subsequently, heavy metal resistant genes that demonstrate strong correlation with concentration of heavy metal are selected as representative genes of respective heavy metal. Homolog q-PCR primers and probes are then designed against these representative genes and validated. As results shown, correlation analysis between heavy metal concentrations and gene frequencies of soil samples from heavy metal contaminated areas (Tainan City and Changhua) City, and soil samples from non contaminated areas (Tainan City and Nantou City) identified chrF (Chromiun), copG (Copper), nikC (Nickel), pbrR (Lead) and zitB/ybgR (Zinc) as the representative genes. Subsequently, among the 25 sets of qPCR probe were designed and validated, 22 sets demonstrated Ct values that are positively correlated to the concentration of heavy metals. In conclusion, this study has successfully shown that Third Generation long read metagenome analysis can greatly shorten the development process of qPCR probes. Moving forward, the accuracy of the probes can be further improved by including more samples in the correlation analysis.

英文關鍵字

Third Generation Metagenome Sequencing, Heavy metal resistance gene profile, Molecular diagnostics