Geosmin是一種臭味物質，存在全世界許多的水庫，造成飲用水的口感和臭味問題。為了建立一套早期預警系統，必須先建立出快速的現地監測方法。即時定量PCR(qPCR)可經由專一性的引子偵測產geosmin基因量之多寡來定量產geosmin之藍綠藻。然而，少有關於將qPCR應用到偵測產geosmin之藍綠藻的研究。因此本研究使用顏博士與Giglio博士新設計針對產geosmin基因的引子與探針進行測試與評估。這些引子與探針以在南澳大利亞分離的26種實驗室培養之產geosmin藍綠藻與不產geosmin藍綠藻進行專一性測試，結果顯示引子與探針都有良好的專一性。接著，引子與探針在SmartCycler® II系統上進行測試，不論使用PCR標準品或純藻DNA都能成功製作出檢量線，其偵測極限可低至100 cells/ml。另外結果也得到一個細胞中至少有一個copy number。
由於引子與探針能成功偵測實驗室培養之產geosmin之藍綠藻，下一步是將其應用到環境樣品。樣品採集地點位於南澳大利亞的Myponga水庫，同時運用傳統的偵測方法(如使用顯微鏡計數產geosmin之藍綠藻細胞數或使用GC/MS偵測樣品中geosmin濃度)和分子生物技術方法(使用即時定量PCR)來偵測含有藻華的水樣。結果顯示傳統的偵測方法與分子生物技術方法有良好的相關性。因此，如搭配快速的DNA萃取法，即時定量PCR的方法具有發展應用潛力且非常適用於現地監測。

Geosmin is a worldwide odor compound in water reservoir that causes taste and odor problems in drinking water. In order to set up an early warning system, a rapid on-site monitoring method for geosmin-producers is necessary. Quantitative real-time PCR (qPCR) is useful for quantifying geosmin-producer by focusing on the specific gene encoding geosmin synthetase. However, there is little report about the qPCR approach which is applied to geosmin-producer. Therefore, several primers and probes designed by Dr. H.K. Yen and Dr. Steven Giglio to detect geosmin synthetase gene in cyanobacteria were evaluated and tested in this study. Each primer and probe sets were tested specificity using 26 different species or strains of laboratory cultured geosmin-producers and non geosmin-producers isolated in South Australia. The results showed that all primers and probes had good specificity. Then, the designed primers and probes were tested on the SmartCycler® II System. Standard curves were built successfully by using PCR standard and pure culture DNA. The detection limit could be low to 102 cells/mL. The results suggested that there was at least one copy in a cell.
Since the developed method worked well on laboratory cultured geosmin-producer, the next step was to apply it to environmental samples. Traditional methods (cell count by microscope and geosmin concentration by GC/MS analysis) and molecular method (qPCR by using Yen’s and Gigilio’s primers and probes) were used at the same time to detect cyanobacteria bloom in Myponga Reservoir (South Australia). The results showed good correlation between molecular method and traditional methods. Therefore, with a rapid DNA extraction method, qPCR would be applicable to on-site monitoring.

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Other:
Instruction of SYBR® Premix Ex TaqTM (Perfect Real Time), Takara bio. Inc.
SmartNote 6.4, Intercalating dye assays on the smartcycler®Ⅱ system,

Internet information:
Cepheid technical support. (http://www.cepheid.com/media/files/smart-notes/SmartNote6.4.pdf)