建立側向流免疫分析法以快速偵測厭氧氨氧化菌之功能性基因表達量

Development of Lateral Flow Immunoassay for Rapid Detection of Functional Gene Expression of Anammox Bacteria

盧昶竹(Chang-Zhu Lu)

指導教授 ： 吳瑞璋

共同指導教授 ： 黃郁慈(Yu-Tzu Huang)

中原大學/工學院/化學工程研究所/碩士(2021年)

https://doi.org/10.6840/cycu202100498

摘要

為了能判定活性污泥中厭氧氨氧化菌的生長情況，根據資料庫中厭氧氨氧化菌的基因序列，本研究針對hzsA基因體設計專一性引子，進行PCR分析各處理系統污泥之厭氧氨氧化菌，以瞭解各系統中所包含的厭氧氨氧化菌種之基因表達量。本研究也利用側向流免疫分析法取代凝膠電泳分析，建立一套簡易的厭氧氨氧化菌檢測技術，具有方便、低成本和檢測時間短等優點，不需要儀器設備，肉眼即可判讀分析結果。
側向流免疫分析法 (LFIA)對 Anammoxoglobus propionicus 和 Kuenenia stuttgartiensis 具有特異性，在其他非目標樣品中未觀察到交叉反應。其偵測極限分別為 0.1 pg 和 0.01 pg。
本研究建立了一種簡單、快速的厭氧氨氧化菌hzsA基因檢測試條。PCR產物可在5分鐘內於試條上顯示結果，使用此視覺化的感測準則可在真實樣品中成功進行基因相對定量分析。

關鍵字

厭氧氨氧化菌 ； 側向流免疫分析法 ； 快速偵測

並列摘要

In order to determine the growth conditions of anammox bacteria in activated sludge, this study designs a specific primer for hzsA gene according to its gene sequence in the database. The target gene underwent a PCR amplification to show its gene expression of anammox bacteria in water-treatment systems.
A simple detection approach of anammox was also developed to use lateral flow immunoassay (LFIA) instead of gel electrophoresis analysis. The LFIA has advantages of convenience, low cost and short detection time. Its analysis results can also be read by naked eyes.
Our LFIA tests had results specific to Anammoxoglobus propionicus and Kuenenia stuttgartiensis. There were no cross-reactions observed to other non-target samples. The detection limit was 0.1 pg and 0.01pg for Anammoxoglobus propionicus and Kuenenia stuttgartiensis, respectively.
In this study, a simple and rapid test format for detection of anammox bacteria hzsA gene was developed. Its PCR product was able to be detected on LFIA strips within 5 minutes. The tests sampled from activated sludge were also successfully perform relative quantitative analysis of genes with this visualized sensing protocol.

並列關鍵字

anammox bacteria ； lateral flow immunoassay ； rapid detection

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