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。為了建立一套預警系統，必須先建立出快速的現地監測方法。雖然即時定量PCR(qPCR)可用來偵測產geosmin之微生物，但因複雜的實驗室DNA萃取程序使其不容易運用於現地。為執行現地偵測，樣本前處理方法必須要簡單、方便、還需具高DNA回收率。本研究將針對288A 引子qPCR條件，細胞收集(以離心法及濾膜過濾法)和細胞破壞(微波破壞法及磁珠震盪法)進行樣品前處理時效之研究探討比較。這些方法以實驗室培養之產geosmin藍綠藻-Anabaena circinalis進行測試。在初裂解時間240秒以上，引子接合溫度47oC，搭配引子濃度0.4μM下有較佳的qPCR放大效率。濾膜過濾法使用聚碳酸酯膜(polycarbonate)比起聚偏氟乙烯纖維膜(PVDF)有較高的細胞回收率 (85±5%V.S 65±5%)然而離心法變異較大 (50±15%)。儘管微波破壞法和磁珠震盪法都可破壞99%以上的細胞。從結果可推測離心-微波法有較穩定的現地qPCR結果，使用濾膜過濾-磁珠震盪法的結果較不穩定。288A引子對Anabaena circinalis的偵測極限約為 5×10^6~5×10^4 cells/ml;使用離心-微波法 偵測極限為 1.0×10^5~1.0×10^4 cells/ml。雖然濾膜過濾-磁珠震盪法的qPCR結果較不穩定，但就快速且高濃縮倍數的優勢來看，此方法較具有發展應用潛力。

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. Although quantitative real-time PCR (qPCR) is useful for detecting geosmin-producer, it is difficult to apply to field testing because of complicated laboratorial DNA extraction method. For on-site monitoring, the sample pretreatment must be simple, rapid, and high efficiency of DNA recovery. In this study, 288A primers designed by Giglio et al. (2008) for qPCR condition optimization, centrifuge and membrane filtration for cell collection, and microwave irradiation and bead-beating for rapid cell lysis were investigated as pretreatment methods. These methods were tested using laboratory cultured geosmin-producer Anabaena circinalis. Primer concentration 0.4μM and annealing temperature 47oC were comparative appropriate condition for qPCR amplification by SmartCycler®Ⅱ System with TaKaRa SYBR® Premix Ex TaqTM. Filtration using polycarbonate membrane showed high recovery of cells (85±5%) compared with those for polyvinyldene fluoride membrane (67±5%) and centrifuge (50±15%). Both microwave irradiation and bead-beating may disrupt >99% cells. Our results implied centrifuge combined with microwave irradiation was able to use as a rapid pretreatment method for on-site qPCR, which was more stable than membrane filtration beads-beating method. The detection limit for 288A primers on Anabaena circinalis genomic DNA was 5×10^6~5×10^4 cells/ml, and detection limit of centrifuge-microwave irradiation was 1.0×10^5~1.0×10^4 cells/ml. Although membrane filtration with bead-beating was not stable at qPCR result, it was a prospective on-site pretreatment method than centrifuge- microwave irradiation method.

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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)

Division cyanophyta kingdom monera.
(http://www.biologie.uni-hamburg.de/b-online/library/webb/BOT311/Cyanobacteria/Cyanobacteria.htm)

Student guide in- result in style. (http://www.studentsguide.in/microbiology/microbiology-index.html)

GE Healthcare, Product for LifeScience, Methods of cell disruption. (http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/Homepage)