藍綠菌廣泛存在於台灣及世界許多淡水水體，藍綠菌代謝物微囊藻毒素(microcystins)、柱孢藻毒素(cylindrospermopsin)及土味物質geosmin對飲用水安全及可口，影響很大。台灣地區水庫水源眾多，且地區分散，發生藍綠菌藻華或代謝物問題時，通常將採集之樣品運送至分析單位，運輸及分析耗時久，對於水庫管理而言緩不濟急。因此，本研究擬建立現地產毒及產臭藍綠菌的快速監測方法，以有效掌握各分散水源中有害藍綠菌之發生潛勢，配合毒素現地監測方法，可以進一步保障民眾用水之安全。
本研究首先開發可同時偵測具產微囊藻毒素(Microcystins)以及具產geosmin之功能基因的分子生物監測方法，研究中應用快速DNA抽取技術、具專一性的引子與探針以及即時定量連鎖反應儀(qPCR)，所開發之技術可在2小時內同時定量水體中該兩種基因的濃度。本研究並將開發之分子生物技術(qPCR)以偵測採集之水樣，同時搭配顯微鏡鏡檢技術、以及酵素免疫學分析方法，用於台灣地區5座水庫現地監測藍綠菌及毒素濃度，並配合實驗室氣相層析質譜儀(GC/MS)分析樣品中geosmin濃度。
研究結果顯示，本研究所建立之現地偵測方法，於現場採樣後2至3小時左右即可得到重要數據，並進一步將數據提供給水庫管理單位，使得各淨水廠有足夠的時間啟動緊急應變之程序，作為水庫藻華、毒素及臭味風險判定參考。研究期間監測中發現4座水庫水源中微囊藻毒素超過1 μg/L問題，後續也分別啟動持續監測採樣或是淨水廠清水採樣，以確保飲用水水質安全。

Prescence of cyanbacterial metabolites in drinking water, such as cyanotoxins and taste and odour (T&O) compounds, may pose health and aesthatic problems to the consumers. Microcystins and cylindrospermopsin are the two most commonly detected cyanotoxin groupds in Taiwan’s reservoirs, and geosmin is among the most detected T&O compounds. Conventionally, water samples potentially contaminated by these cyanobaterial metabolites are transported to and analyzed in the laboratories with sophiscated instruments. It usually requires more than 2 days to obtain analytical results, which might be too late as contaminated water may have entered consumers’ tap. Therefore, it is urgently needed to have a rapid and robust analytical scheme for the detection of cyanobacterial metabolites in drinking water systems.
In this study, a rapid on-site monitoring method for microcystins, cylindrospermopsin and geosmin-producers is developed. Quantitative real-time PCR (qPCR) is employed to quantify the targeted metabolite producers, by detecting the specific gene encoding target synthetase. Specific primers and probes were developed and tested for the targeted genes. The results showed the all the primers and probes developed are very specific to the targeted genes, with successful standard curved constructed between PCR standard and pure culture DNA. The method was then applied in 5 fresh water bodies in Taiwan, together with microscopic cell counting and cyanotoxin and geosmin concentration determination.
The results show that the method established is able tobe used on-site, with key information to be determined with 2-3 hours after sampling. The information may provide a basis for decision making for the managers of reservoirs and utilities. Four of the sampling runs conducted in the reservoirs were found to have microcystin concentration higher than 1 μg/L. To secure safe drinking water, samples for raw water and finished water were also collected for the water treatment plants. Fortunately, all the finished water samples show no contimation of cyanotoxins. This approach may provide a quick basis for the management of cyanobacteria and metabolites in drinkgin water systems.

Ahmad, N. W., P. Dixon, J. A. Shields, H. S. Dua, and L. A. Donoso. "Identification and Rapid Screening of a Drai Rflp by Pcr in the Retinoblastoma Gene (Vol 78, Pg 159, 1994)." British Journal of Ophthalmology 78, no. 12 (1994): 950-950.

Berg, K., W. W. Carmichael, O. M. Skulberg, C. Benestad, and B. Underdal. "Investigation of a Toxic Water-Bloom of Microcystis-Aeruginosa (Cyanophyceae) in Lake-Akersvatn, Norway." Hydrobiologia 144, no. 2 (1987): 97-103.

Binder, B J, and S W Chisholm. "Relationship between DNA Cycle and Growth Rate in Synechococcus Sp. Strain Pcc 6301." Journal of Bacteriology 172, no. 5 (1990): 2313-2319.

Bookstein, R., C. C. Lai, H. To, and W. H. Lee. "Pcr-Based Detection of a Polymorphic Bamhi Site in Intron-1 of the Human Retinoblastoma (Rb) Gene." Nucleic Acids Research 18, no. 6 (1990): 1666-1666.

Boutou, S., and P. Chatonnet. "Rapid Headspace Solid-Phase Microextraction/Gas Chromatographic/Mass Spectrometric Assay for the Quantitative Determination of Some of the Main Odorants Causing Off-Flavours in Wine." Journal of Chromatography A 1141, no. 1 (2007): 1-9.

Bruchet, A., and J. P. Duguet. "Role of Oxidants and Disinfectants on the Removal, Masking and Generation of Tastes and Odours." Water Science and Technology 49, no. 9 (2004): 297-306.

Carmichael, W. W., J. T. Eschedor, G. M. L. Patterson, and R. E. Moore. "Toxicity and Partial Structure of a Hepatotoxic Peptide Produced by the Cyanobacterium Nodularia-Spumigena Mertens Emend L575 from New-Zealand." Applied and Environmental Microbiology 54, no. 9 (1988): 2257-2263.

CHRISTOPHER W.K. CHOW, MARY DRIKAS, JENNY HOUSE, MICHAEL, and RENATE M.A. VELZEBOER D. BURCH "The Impact of Conventional Water Treatment Processes on Cells of the Cyanobacterium Microcystis Aeruginosa." Water Research 33, no. 15 (1999): 3253-3262.

CRC. Blue Green Algae: A Guide., 2004.

Dorigo, U., L. Volatier, and J. F. Humbert. "Molecular Approaches to the Assessment of Biodiversity in Aquatic Microbial Communities." Water Research 39, no. 11 (2005): 2207-2218.

Dzialowski, Andrew R., Val H. Smith, Donald G. Huggins, Frank deNoyelles, Niang-Choo Lim, Debbie S. Baker, and Jason H. Beury. "Development of Predictive Models for Geosmin-Related Taste and Odor in Kansas, USA, Drinking Water Reservoirs." Water Research 43, no. 11 (2009): 2829-2840.

Falconer, Ian R. "Potential Impact on Human Health of Toxic Cyanobacteria." Phycologia 35, no. 6 SUPPL. (1996): 6-11.

Fastner, J., R. Heinze, and I. Chorus. "Microcystin-Content, Hepatotoxicity and Cytotoxicity of Cyanobacteria in Some German Water Bodies." Water Science and Technology 32, no. 4 (1995): 165-170.

Gelder, Russell N. Van. "Applications of the Polymerase Chain Reaction to Diagnosis of Ophthalmic Disease." Survey of Ophthalmology 46, (2001): 248-258.

Gilbride, K. A., D. Y. Lee, and L. A. Beaudette. "Molecular Techniques in Wastewater: Understanding Microbial Communities, Detecting Pathogens, and Real-Time Process Control." Journal of Microbiological Methods 66, no. 1 (2006): 1-20.

Graham, J. L., J. R. Jones, S. B. Jones, J. A. Downing, and T. E. Clevenger. "Environmental Factors Influencing Microcystin Distribution and Concentration in the Midwestern United States." Water Research 38, no. 20 (2004): 4395-4404.

Haider, S., V. Naithani, P. N. Viswanathan, and P. Kakkar. "Cyanobacterial Toxins: A Growing Environmental Concern." Chemosphere 52, no. 1 (2003): 1-21.

Harding, William R., and Susan Wright. "Initial Findings Regarding Changes in Phyto- and Zooplankton Composition and Abundance Following the Temporary Drawdown and Refilling of a Shallow, Hypertrophic South African Coastal Lake." Lake and Reservoir Management 15, no. 1 (1999): 47-53.

Hernandez, M., M. Macia, C. Padilla, and F. F. Del Campo. "Modulation of Human Polymorphonuclear Leukocyte Adherence by Cyanopeptide Toxins." Environmental Research 84, no. 1 (2000): 64-68.

Hitzfeld, B. C., S. J. Hoger, and D. R. Dietrich. "Cyanobacterial Toxins: Removal During Drinking Water Treatment, and Human Risk Assessment." Environmental Health Perspectives 108, (2000): 113-122.

Hummert, C., J. Dahlmann, M. Reichelt, and B. Luckas. "Analytical Techniques for Monitoring Harmful Cyanobacteria in Lakes." Lakes and Reservoirs Research and Management 6, no. 2 (2001): 159-168.

Hung-Kai Yen, Tsair-Fuh Lin, Pao-Chi Liao. "Simultaneous Detection of Nine Cyanotoxins in Drinking Water Using Dual Solid-Phase Extraction and Liquid Chromatography–Mass Spectrometry." Toxicon 58, no. 2 (2011): 209-218.

Hyeon, Ji-Yeon, Chankyu Park, In-Soo Choi, Peter S. Holt, and Kun-Ho Seo. "Development of Multiplex Real-Time Pcr with Internal Amplification Control for
Simultaneous Detection of Salmonella and Cronobacter in Powdered Infant Formula." International Journal of Food Microbiology, (2010).

Izaguirre, G., C. J. Hwang, S. W. Krasner, and M. J. McGuire. "Geosmin and 2-Methylisoborneol from Cyanobacteria in 3 Water-Supply Systems." Applied and Environmental Microbiology 43, no. 3 (1982): 708-714.

Izaguirre, G., and W. D. Taylor. "Geosmin and Mib Events in a New Reservoir in Southern California." Water Science and Technology 55, no. 5 (2007): 9-14.

Jähnichen, Sabine, Kathrin Jäschke, Falk Wieland, Gabriele Packroff, and Jürgen Benndorf. "Spatio-Temporal Distribution of Cell-Bound and Dissolved Geosmin in Wahnbach Reservoir: Causes and Potential Odour Nuisances in Raw Water." Water Research 45, no. 16 (2011): 4973-4982.

J.P. Rasmussen, S. Giglio, P.T. Monis, R.J. Campbell and C.P. Saint. "Development and Field Testing of a Real-Time Pcr Assay for
Cylindrospermopsin-Producing Cyanobacteria." Journal of Applied Microbiology 104, (2008): 1503–1515.

Jamal Al-Tebrineh, Leanne A. Pearson, Serhat A. Yasar, Brett A. Neilan. "A Multiplex Qpcr Targeting Hepato- and Neurotoxigenic Cyanobacteria of Global Significance." Harmful Algae, no. 15 (2012): 19-25.

Johansson, M. K., H. Fidder, D. Dick, and R. M. Cook. "Intramolecular Dimers: A New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes." Journal of the American Chemical Society 124, no. 24 (2002): 6950-6956.

Juttner, F., and S. B. Watson. "Biochemical and Ecological Control of Geosmin and 2-Methylisoborneol in Source Waters." Appl Environ Microbiol 73, no. 14 (2007): 4395-406.

Kaneko, Takakazu, Nobuyoshi Nakajima, Shinobu Okamoto, Iwane Suzuki, Yuuhiko Tanabe, Masanori Tamaoki, Yasukazu Nakamura, Fumie Kasai, Akiko Watanabe, Kumiko Kawashima, Yoshie Kishida, Akiko Ono, Yoshimi Shimizu, Chika Takahashi, Chiharu Minami, Tsunakazu Fujishiro, Mitsuyo Kohara, Midori Katoh, Naomi Nakazaki, Shinobu Nakayama, Manabu Yamada, Satoshi Tabata, and Makoto M. Watanabe. "Complete Genomic Structure of the Bloom-Forming Toxic Cyanobacterium Microcystis Aeruginosa Nies-843." DNA Research 14, no. 6 (2007): 247-256.

Krasner, S. W., C. J. Hwang, and M. J. McGuire. "A Standard Method for Quantification of Earthy-Musty Odorants in Water, Sediments, and Algal Cultures." Water Science and Technology 15, no. 6-7 (1983): 127-138.

Lalezary, S., M. Pirbazari, and M. J. McGuire. "Oxidation of Five Earthy-Musty Taste and Odor Compounds." American Water Works Association Journal 78, no. 3 (1986): 62-69.

Leutenegger, C. M., D. Klein, R. Hofmann-Lehmann, C. Mislin, U. Hummel, J. Boni, F. Boretti, W. H. Guenzburg, and H. Lutz. "Rapid Feline Immunodeficiency Virus Provirus Quantitation by Polymerase Chain Reaction Using the Taqman (R) Fluorogenic Real-Time Detection System." Journal of Virological Methods 78, no. 1-2 (1999): 105-116.

Lloyd, S. W., J. M. Lea, P. V. Zimba, and C. C. Grimm. "Rapid Analysis of Geosmin and 2-Methylisoborneol in Water Using Solid Phase Micro Extraction Procedures." Water Research 32, no. 7 (1998): 2140-2146.

Maatouk, I., N. Bouaicha, D. Fontan, and Y. Levi. "Seasonal Variation of Microcystin Concentrations in the Saint-Caprais Reservoir (France) and Their Removal in a Small Full-Scale Treatment Plant." Water Research 36, no. 11 (2002): 2891-2897.

Maizels, M., and W. L. Budde. "A Lc/Ms Method for the Determination of Cyanobacteria Toxins in Water." Analytical Chemistry 76, no. 5 (2004): 1342-1351.

Martin L Saker, James S Metcalf, Geoffrey A Codd, Vitor M Vasconcelos. "Accumulation and Depuration of the Cyanobacterial Toxin Cylindrospermopsin in the Freshwater Mussel Anodonta Cygnea." Toxicon 43, no. 2 (2004): 185-194.

Mastrangelo, D., N. Squitieri, S. Bruni, T. Hadjistilianou, and R. Frezzotti. "The Polymerase Chain Reaction (Pcr) in the Routine Genetic Characterization of Retinoblastoma: A Tool for the Clinical Laboratory." Survey of Ophthalmology 41, no. 4 (1997): 331-340.

Michinaka A., Yen H.K., Chiu Y.T., Tsao H.W., Lin T.F. "Rapid on-Site Multiplex Assays for Total and Toxigenic Microcystis Using Real-Time Pcr with Microwave Cell Disruption." Water Sci. Technol., (2012).

Mikalsen, B., G. Boison, O. M. Skulberg, J. Fastner, W. Davies, T. M. Gabrielsen, K. Rudi, and K. S. Jakobsen. "Natural Variation in the Microcystin Synthetase Operon Mcyabc and Impact on Microcystin Production in Microcystis Strains." Journal of Bacteriology 185, no. 9 (2003): 2774-2785.

Moreira, B. G., Y. You, M. A. Behlke, and R. Owczarzy. "Effects of Fluorescent Dyes, Quenchers, and Dangling Ends on DNA Duplex Stability." Biochemical and Biophysical Research Communications 327, no. 2 (2005): 473-484.

Nicolaisen, K., V. Mariscal, R. Bredemeier, R. Pernil, S. Moslavac, R. Lopez-Igual, I. Maldener, A. Herrero, E. Schleiff, and E. Flores. "The Outer Membrane of a Heterocyst-Forming Cyanobacterium Is a Permeability Barrier for Uptake of Metabolites That Are Exchanged between Cells." Molecular Microbiology 74, no. 1 (2009): 58-70.

Nilsson, T., T. O. Larsen, L. Montanarella, and J. O. Madsen. "Application of Head-Space Solid-Phase Microextraction for the Analysis of Volatile Metabolites Emitted by Penicillium Species." Journal of Microbiological Methods 25, no. 3 (1996): 245-255.

Onadim, Z., and J. K. Cowell. "Application of Pcr Amplification of DNA from Paraffin Embedded Tissue-Sections to Linkage Analysis in Familial Retinoblastoma." Journal of Medical Genetics 28, no. 5 (1991): 312-316.

Pawliszyn, J. Applications of Solid Phase Microextraction. . UK: The Royal Society of Chemistry., 1999.

Persson, P. E. "Sensory Properties and Analysis of 2 Muddy Odor Compounds, Geosmin and 2-Methylisoborneol, in Water and Fish." Water Research 14, no. 8 (1980): 1113-1118.

________. "Off-Flavors in Aquatic Ecosystems - an Introduction." Water Science and Technology 15, no. 6-7 (1983): 1-11.

Pouria, S., A. de Andrade, J. Barbosa, R. L. Cavalcanti, V. T. S. Barreto, C. J. Ward, W. Preiser, G. K. Poon, G. H. Neild, and G. A. Codd. "Fatal Microcystin Intoxication in Haemodialysis Unit in Caruaru, Brazil." Lancet 352, no. 9121 (1998): 21-26.

Pyo, D., and H. Shin. "Extraction and Analysis of Microcystins Rr and Lr in Cyanobacteria Using a Cyano Cartridge." Journal of Biochemical and Biophysical Methods 51, no. 2 (2002): 103-109.

Rantala, A., P. Rajaniemi-Wacklin, C. Lyra, L. Lepisto, J. Rintala, J. Mankiewiez-Boczek, and K. Sivonen. "Detection of Microcystin-Producing Cyanobacteria in Finnish Lakes with Genus-Specific Microcystin Synthetase Gene E (Mcye) Pcr and Associations with Environmental Factors." Applied and Environmental Microbiology 72, no. 9 (2006): 6101-6110.

Rasmussen, J. P., S. Giglio, P. T. Monis, R. J. Campbell, and C. P. Saint. "Development and Field Testing of a Real-Time Pcr Assay for Cylindrospermopsin-Producing Cyanobacteria." Journal of Applied Microbiology 104, no. 5 (2008): 1503-1515.

Rinta-Kanto, J. M., A. J. A. Ouellette, G. L. Boyer, M. R. Twiss, T. B. Bridgeman, and S. W. Wilhelm. "Quantification of Toxic Microcystis Spp. During the 2003 and 2004 Blooms in Western Lake Erie Using Quantitative Real-Time Pcr." Environmental Science & Technology 39, no. 11 (2005): 4198-4205.

Rippka, R. "Isolation and Purification of Cyanobacteria." Methods in Enzymology 167, (1988): 3-27.

Robarts, R. D., and T. Zohary. "Temperature Effects on Photosynthetic Capacity, Respiration, and Growth-Rates of Bloom-Forming Cyanobacteria." New Zealand Journal of Marine and Freshwater Research 21, no. 3 (1987): 391-399.

Roberts, Thomas M., Lynn C. Klotz, and Alfred R. Loeblich Iii. "Characterization of a Blue-Green Algal Genome." Journal of Molecular Biology 110, no. 2 (1977): 341-361.

Rouhiainen, L., T. Vakkilainen, B. L. Siemer, W. Buikema, R. Haselkorn, and K. Sivonen. "Genes Coding for Hepatotoxic Heptapeptides (Microcystins) in the Cyanobacterium Anabaena Strain 90." Applied and Environmental Microbiology 70, no. 2 (2004): 686-692.

Stanier, R. Y., and G. Cohenbazire. "Phototropic Prokaryotes - Cyanobacteria." Annual Review of Microbiology 31, (1977): 225-274.

Sunda, W. G. "Trace Metal Interactions with Marine Phytoplankton." Biological Oceanography 6, no. 5-6 (1991): 411-442.

Thurnheer, T., R. Gmur, and B. Guggenheim. "Multiplex Fish Analysis of a Six-Species Bacterial Biofilm." Journal of Microbiological Methods 56, no. 1 (2004): 37-47.

Tobler, Nadia E., Monika Pfunder, Katrin Herzog, Juerg E. Frey, and Martin Altwegg. "Rapid Detection and Species Identification of Mycobacterium Spp. Using Real-Time Pcr and DNA-Microarray." Microbiological Methods 66, (2006): 116-124.

USEPA. Creating a Cyanotoxin Target List for the Unregulated Contaminant Monitoring Rule., 2001.

Uwins, H. K., P. Teasdale, and H. Stratton. "A Case Study Investigating the Occurrence of Geosmin and 2-Methylisoborneol (Mib) in the Surface Waters of the Hinze Dam, Gold Coast, Australia." Water Science and Technology 55, no. 5 (2007): 231-238.

Vaitomaa, Jaana, Anne Rantala, Katrianna Halinen, Leo Rouhiainen, Petra Tallberg, Lena Mokelke, and Kaarina Sivonen. "Quantitative Real-Time Pcr for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes." Applied and Environmental Microbiology 69, no. 12 (2003): 7289-7297.

Valasek, M. A., and J. J. Repa. "The Power of Real-Time Pcr." Advances in Physiology Education 29, no. 3 (2005): 151-159.

Van Der Westhuizen, A. J., and J. N. Eloff. "Effect of Temperature and Light on the Toxicity and Growth of the Blue-Green Alga Microcystis-Aeruginosa Uv-006." Planta (Heidelberg) 163, no. 1 (1985): 55-59.

Vasas, G., A. Gaspar, C. Pager, G. Suranyi, C. Mathe, M. M. Hamvas, and G. Borbely. "Analysis of Cyanobacterial Toxins (Anatoxin-a, Cylindrospermopsin, Microcystin-Lr) by Capillary Electrophoresis." Electrophoresis 25, no. 1 (2004): 108-115.

W., Carmichael W. "Assessment of Blue-Green Algal Toxins in Raw and Finished Drinking Water." AWWA Research Foundation., (2001).

Welker, M., L. Sejnohova, D. Nemethova, H. von Dohren, J. Jarkovsky, and B. Marsalek. "Seasonal Shifts in Chemotype Composition of Microcystis Sp Communities in the Pelagial and the Sediment of a Shallow Reservoir." Limnology and Oceanography 52, no. 2 (2007): 609-619.

Wen-Hsing Hsieh, Wei-Nung Hung, Gen-Shuh Wang, Shu-Ting Hsieh and Tsair-Fuh Lin. "Effect of Ph on the Analysis of 2-Mib and Geosmin in Water." Water Air Soil Pollut 223, (2012): 715-721.

Xie, L. Q., P. Xie, S. X. Li, H. J. Tang, and H. Liu. "The Low Tn : Tp Ratio, a Cause or a Result of Microcystis Blooms?" Water Research 37, no. 9 (2003): 2073-2080.

Young, W. F., H. Horth, R. Crane, T. Ogden, and M. Arnott. "Taste and Odour Threshold Concentrations of Potential Potable Water Contaminants." Water Research 30, no. 2 (1996): 331-340.

Zentilin, L., and M. Giacca. "Competitive Pcr for Precise Nucleic Acid Quantification." Nature Protocols 2, no. 9 (2007): 2092-2104.

Zhang, T., and H. H. P. Fang. "Applications of Real-Time Polymerase Chain Reaction for Quantification of Microorganisms in Environmental Samples." Applied Microbiology and Biotechnology 70, no. 3 (2006): 281-289.

曹翔崴，(2011)，水庫中Geosmin產生源分子生物定量方法之開發與應用，國立成功大學環境工程學系碩士論文。
邱宜亭，(2009)，高錳酸鉀對三種藍綠菌破壞及其代謝物釋出之研究，國立成功大學環境工程學系碩士論文。
張哲源，(2010)，開發SYBR Green Real-time PCR 現地偵測Geosmin產臭基因之方法，國立成功大學環境工程學系碩士論文。