柱孢藻毒素(Cylindrospermopsin,CYN)是一種常見的藍綠細菌有害二次代謝物，其結構穩定且具有高水溶性，在淨水處理程序中難以被去除，且會對人體造成許多負面影響。台灣本島水庫與金門地區水庫都曾發現過柱孢藻毒素的存在，顯示柱孢藻毒素對台灣飲用水體具有潛在的危害，相關應對措施的提出及預警策略的建立是必要的。因此本研究擬以即時定量連鎖聚合酶反應(qPCR) 之Taqman系統建立總柱孢藻細胞與產CYN細胞之現地偵測技術，以期可即刻掌握柱孢藻毒素對飲用水體的潛在危害，降低飲用水受到污染所造成的可能危害，進一步保障民眾用水安全。
　　本研究成功地以qPCR TaqMan系統建立偵測產CYN細胞功能性基因 (pks)與總柱孢藻細胞功能性基因(rpoC1)之單一定量與雙重定量分析技術，此外，本研究亦同時以酵素連結免疫分析法(ELISA)進行柱孢藻毒素濃度測量，和以顯微鏡細胞計數測量總柱孢藻細胞數，並進行三者間之相關性分析，整體分析時間僅需2~3小時。本研究將此現地偵測技術應用於台灣本島共11座水庫(共73個採樣點)和金門地區共11水庫與2座淨水場(共60個採樣點)，結果顯示，總柱孢藻細胞偵測基因(rpoC1)定量結果與顯微鏡細胞計數之數據間存在良好之正相關性；而產CYN細胞偵測基因(pks)定量結果與ELISA分析藻毒濃度雖無濃度趨勢上的相關性，但在60筆金門地區水庫分析結果中，有九成的數據可觀察到兩者間在水體中存在與否的一致性。另一方面，本研究中亦針對qPCR雙重定量系統進行分析條件的最適化，來改善因兩偵測基因間影響造成螢光檢測值偏低的情形，以期未來能將此技術廣泛應用於各種類型水體中總柱孢藻細胞與產CYN細胞的監測。

Cylindrospermopsin (CYN) is a toxic secondary metabolite produced by Cyanobacteria in water body. Because of its stable structure and high solubility in water, CYN is not easy to remove by conventional drinking water treatment processes, resulting in negative effect on human health. CYN has been discovered in a few of Taiwan’s reservoirs, including in main island and in Kinmen island. Therefore, a method to accurately and rapidly estimate the risk associated with the presence of CYN in drinking water sources in required for the protection of public health. Therefore, in this study we proposed to establish an on-site monitoring technology for Cylindrospermopsis cells and CYN-producing cells using qPCR combined with TaqMan system.
Both single and dual quantification systems targeted for the functional genes of Cylindrospermopsis cells, rpoC1, and CYN producing cells, pks, using qPCR combined with TaqMan system were successfully established. The qPCR-TaqMan method was further applied in the on-site monitoring of the targeted genes present in Taiwan’s reservoirs. A total of 73 and 60 samples were collected and analyzed for 11 reservoirs in main island and 11 reservoirs and 2 drinking-water treatment systems in Kingmen island, respectively. During the monitoring, an enzyme-linked immunosorbent assay (ELISA) and an optical microscope were also employed to analyze the concentrations of CYN and Cylindrospermopsis cells, respectively.
Monitoring results showed that good correlations between rpoC1 gene copy numbers detected with the qPCR method and cell numbers enumerated with optical microscope. For another targeted gene, pks, although no correlations were obtained, 90% of the Kinmen samples showed co-occurrence of pks gene copies and CYN concentrations measured with ELISA. A dual quantification system for both pks and rpoC1 genes was also developed. The experimental parameters were tuned to improve the detection of both genes, serving as a basis to better apply the dual detection system in the future.

Griffiths, D.J. and Saker, M.L. (2003) The Palm Island mystery disease 20 years on: a review of research on the cyanotoxin cylindrospermopsin. Environmental Toxicology 18(2), 78-93.
Moreira, C., Azevedo, J., Antunes, A. and Vasconcelos, V. (2012) Cylindrospermopsin: occurrence, methods of detection and toxicology. J Appl Microbiol.
Kinnear, S. (2010) Cylindrospermopsin: a decade of progress on bioaccumulation research. Mar Drugs 8(3), 542-564.
Hitzfeld, B.C., Höger, S.J. and Dietrich, D.R. (2000) Cyanobacterial toxins: removal during drinking water treatment, and human risk assessment. Environmental Health Perspectives 108(Suppl 1), 113.
Newcombe, G., House, J., Ho, L., Baker, P. and Burch, M. (2010) Management strategies for cyanobacteria (blue-green algae): A guide for water utilities, WQRA.
林財富 與曾怡禎 (2007) 飲用水水源及水質中產毒藻種及藻類毒素之研究。.行政院環境保護署。
Schembri, M.A., Neilan, B.A. and Saint, C.P. (2001) Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii. Environmental Toxicology 16(5), 413-421.
Kaebernick, M. and Neilan, B.A. (2001) Ecological and molecular investigations of cyanotoxin production. FEMS microbiology ecology 35(1), 1-9.
Falconer, I.R. and Humpage, A.R. (2005) Health risk assessment of cyanobacterial (blue-green algal) toxins in drinking water. International Journal of Environmental Research and Public Health 2(1), 43-50.
Antoniou, M.G., De la Cruz, A.A. and Dionysiou, D.D. (2005) Cyanotoxins: New generation of water contaminants. Journal of environmental engineering 131(9), 1239-1243.
Funari, E. and Testai, E. (2008) Human health risk assessment related to cyanotoxins exposure. Crit Rev Toxicol 38(2), 97-125.
USEPA (2001) Creating a Cyanotoxin Target List for the Unregulated Contaminant Monitoring Rule.
Chorus, E.I. and Bartram, J. (1999) Toxic cyanobacteria in water: a guide to their public health consequences, monitoring and management.
Ohtani, I., Moore, R.E. and Runnegar, M.T. (1992) Cylindrospermopsin: a potent hepatotoxin from the blue-green alga Cylindrospermopsis raciborskii. Journal of the American Chemical Society 114(20), 7941-7942.
Pearson, L., Mihali, T., Moffitt, M., Kellmann, R. and Neilan, B. (2010) On the chemistry, toxicology and genetics of the cyanobacterial toxins, microcystin, nodularin, saxitoxin and cylindrospermopsin. Mar Drugs 8(5), 1650-1680.
Chiswell, R.K., Shaw, G.R., Eaglesham, G., Smith, M.J., Norris, R.L., Seawright, A.A. and Moore, M.R. (1999) Stability of cylindrospermopsin, the toxin from the cyanobacterium, Cylindrospermopsis raciborskii: Effect of pH, temperature, and sunlight on decomposition. Environmental Toxicology 14(1), 155-161.
Falconer, I.R. (2005) Cyanobacteria‐Toxins in Drinking Water, Wiley Online Library.
USEPA (2006) Toxicological Review of Cyanobacterial Toxins: Cylindrospermopsin (External Review Draft). United States Environmental Protection Agency.
Runnegar, M.T., Xie, C., Snider, B.B., Wallace, G.A., Weinreb, S.M. and Kuhlenkamp, J. (2002) In vitro hepatotoxicity of the cyanobacterial alkaloid cylindrospermopsin and related synthetic analogues. Toxicological Sciences 67(1), 81-87.
Banker, R., Carmeli, S., Werman, M., Teltsch, B., Porat, R. and Sukenik, A. (2001) Uracil moiety is required for toxicity of the cyanobacterial hepatotoxin cylindrospermopsin. Journal of Toxicology and Environmental Health Part A 62(4), 281-288.
Looper, R.E., Runnegar, M.T. and Williams, R.M. (2005) Synthesis of the putative structure of 7-deoxycylindrospermopsin: C7 oxygenation is not required for the inhibition of protein synthesis. Angew Chem Int Ed Engl 44(25), 3879-3881.
Mazmouz, R., Chapuis-Hugon, F., Mann, S., Pichon, V., Mejean, A. and Ploux, O. (2010) Biosynthesis of cylindrospermopsin and 7-epicylindrospermopsin in Oscillatoria sp. strain PCC 6506: identification of the cyr gene cluster and toxin analysis. Appl Environ Microbiol 76(15), 4943-4949.
Norris, R.L., Eaglesham, G.K., Pierens, G., Shaw, G.R., Smith, M.J., Chiswell, R.K., Seawright, A.A. and Moore, M.R. (1999) Deoxycylindrospermopsin, an analog of cylindrospermopsin from Cylindrospermopsis raciborskii. Environmental Toxicology 14(1), 163-165.
Li, R., Carmichael, W.W., Brittain, S., Eaglesham, G.K., Shaw, G.R., Liu, Y. and Watanabe, M.M. (2001a) First report of the cyanotoxins cylindrospermopsin and deoxycylindrospermopsin from Raphidiopsis curvata (Cyanobacteria). Journal of Phycology 37(6), 1121-1126.
Seifert, M., McGregor, G., Eaglesham, G., Wickramasinghe, W. and Shaw, G. (2007) First evidence for the production of cylindrospermopsin and deoxy-cylindrospermopsin by the freshwater benthic cyanobacterium,Lyngbya wollei(Farlow ex Gomont) Speziale and Dyck. Harmful Algae 6(1), 73-80.
Banker, R., Teltsch, B., Sukenik, A. and Carmeli, S. (2000) 7-Epicylindrospermopsin, a Toxic Minor Metabolite of the Cyanobacterium Aphanizomenon o valisporum from Lake Kinneret, Israel. Journal of natural products 63(3), 387-389.
Westrick, J.A., Szlag, D.C., Southwell, B.J. and Sinclair, J. (2010) A review of cyanobacteria and cyanotoxins removal/inactivation in drinking water treatment. Analytical and bioanalytical chemistry 397(5), 1705-1714.
Jiang, Y., Xiao, P., Yu, G., Sano, T., Pan, Q. and Li, R. (2012) Molecular basis and phylogenetic implications of deoxycylindrospermopsin biosynthesis in the cyanobacterium Raphidiopsis curvata. Appl Environ Microbiol 78(7), 2256-2263.
Li, R., Carmichael, W., Brittain, S., Eaglesham, G., Shaw, G., Mahakhant, A., Noparatnaraporn, N., Yongmanitchai, W., Kaya, K. and Watanabe, M. (2001b) Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria). Toxicon 39(7), 973-980.
Preußel, K., Stüken, A., Wiedner, C., Chorus, I. and Fastner, J. (2006) First report on cylindrospermopsin producing Aphanizomenon flos-aquae (Cyanobacteria) isolated from two German lakes. Toxicon 47(2), 156-162.
Banker, R., Carmeli, S., Hadas, O., Teltsch, B., Porat, R. and Sukenik, A. (1997) IDENTIFICATION OF CYLINDROSPERMOPSIN IN APHANIZOMENON OVALISPORUM (CYANOPHYCEAE) ISOLATED FROM LAKE KINNERET, ISRAEL1. Journal of Phycology 33(4), 613-616.
Spoof, L., Berg, K.A., Rapala, J., Lahti, K., Lepistö, L., Metcalf, J.S., Codd, G.A. and Meriluoto, J. (2006) First observation of cylindrospermopsin in Anabaena lapponica isolated from the boreal environment (Finland). Environmental toxicology 21(6), 552-560.
Harada, K.-i., Ohtani, I., Iwamoto, K., Suzuki, M., Watanabe, M.F., Watanabe, M. and Terao, K. (1994) Isolation of cylindrospermopsin from a cyanobacterium Umezakia natans and its screening method. Toxicon 32(1), 73-84.
Rücker, J., Stüken, A., Nixdorf, B., Fastner, J., Chorus, I. and Wiedner, C. (2007) Concentrations of particulate and dissolved cylindrospermopsin in 21 Aphanizomenon-dominated temperate lakes. Toxicon 50(6), 800-809.
Bittencourt-Oliveira, M.d.C. (2011) Cylindrospermopsin in Water Supply Reservoirs in Brazil Determined by Immunochemical and Molecular Methods. Journal of Water Resource and Protection 03(06), 349-355.
Alster, A., Kaplan-Levy, R.N., Sukenik, A. and Zohary, T. (2010) Morphology and phylogeny of a non-toxic invasive Cylindrospermopsis raciborskii from a Mediterranean Lake. Hydrobiologia 639(1), 115-128.
Chonudomkul, D., Yongmanitchai, W., Theeragool, G., Kawachi, M., Kasai, F., Kaya, K. and Watanabe, M.M. (2004) Morphology, genetic diversity, temperature tolerance and toxicity of Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) strains from Thailand and Japan. FEMS microbiology ecology 48(3), 345-355.
Wilson, K.M., Schembri, M.A., Baker, P.D. and Saint, C.P. (2000) Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR. Applied and Environmental Microbiology 66(1), 332-338.
Stuken, A., Campbell, R.J., Quesada, A., Sukenik, A., Dadheech, P.K. and Wiedner, C. (2009) Genetic and morphologic characterization of four putative cylindrospermopsin producing species of the cyanobacterial genera Anabaena and Aphanizomenon. Journal of Plankton Research 31(5), 465-480.
Wu, Z., Shi, J., Xiao, P., Liu, Y. and Li, R. (2011) Phylogenetic analysis of two cyanobacterial genera Cylindrospermopsis and Raphidiopsis based on multi-gene sequences. Harmful Algae 10(5), 419-425.
Niiyama, Y., Tuji, A. and Tsujimura, S. (2011) Umezakia natans M. Watan. does not belong to Stigonemataceae but to Nostocaceae. Fottea 11, 163-169.
Fergusson, K.M. and Saint, C.P. (2003) Multiplex PCR assay for Cylindrospermopsis raciborskii and cylindrospermopsin-producing cyanobacteria. Environ Toxicol 18(2), 120-125.
Yılmaz, M., Phlips, E.J., Szabo, N.J. and Badylak, S. (2008) A comparative study of Florida strains of Cylindrospermopsis and Aphanizomenon for cylindrospermopsin production. Toxicon 51(1), 130-139.
張雅雯 (2005) 柱孢藻在金門水庫形成優勢之原因探討. 臺灣大學植物科學研究所學位論文 (2005 年).
Barrios-Llerena, M.E., Burja, A.M. and Wright, P.C. (2007) Genetic analysis of polyketide synthase and peptide synthetase genes in cyanobacteria as a mining tool for secondary metabolites. Journal of industrial microbiology & biotechnology 34(6), 443-456.
Shalev‐Alon, G., Sukenik, A., Livnah, O., Schwarz, R. and Kaplan, A. (2002) A novel gene encoding amidinotransferase in the cylindrospermopsin producing cyanobacterium Aphanizomenon ovalisporum. FEMS microbiology letters 209(1), 87-91.
Mihali, T.K., Kellmann, R., Muenchhoff, J., Barrow, K.D. and Neilan, B.A. (2008) Characterization of the gene cluster responsible for cylindrospermopsin biosynthesis. Applied and Environmental Microbiology 74(3), 716-722.
Saker, M.L. and Neilan, B.A. (2001) Varied diazotrophies, morphologies, and toxicities of genetically similar isolates of Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) from northern Australia. Applied and Environmental Microbiology 67(4), 1839-1845.
Dyble, J., Tester, P. and Litaker, R. (2006) Effects of light intensity on cylindrospermopsin production in the cyanobacterial HAB species Cylindrospermopsis raciborskii. African Journal of Marine Science 28(2), 309-312.
Saker, M.L. and Griffiths, D.J. (2000) The effect of temperature on growth and cylindrospermopsin content of seven isolates of Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) from water bodies in northern Australia. Phycologia 39(4), 349-354.
Hawkins, P.R., Putt, E., Falconer, I. and Humpage, A. (2001) Phenotypical variation in a toxic strain of the phytoplankter, Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) during batch culture. Environmental Toxicology 16(6), 460-467.
Ramos M. , Y. (2011) 發展產柱孢藻毒素藍綠菌快速監測技術之研究。林財富教授。國立成功大學環境工程系碩士學位論文。
Berry, J.P. and Lind, O. (2010) First evidence of “paralytic shellfish toxins” and cylindrospermopsin in a Mexican freshwater system, Lago Catemaco, and apparent bioaccumulation of the toxins in “tegogolo” snails (Pomacea patula catemacensis). Toxicon 55(5), 930-938.
Mastrangelo, D., Squitieri, N., Bruni, S., Hadjistilianou, T. and Frezzotti, R. (1997) The polymerase chain reaction (PCR) in the routine genetic characterization of retinoblastoma: A tool for the clinical laboratory. Survey of Ophthalmology 41(4), 331-340.
Ahmad, N.N., Dixon, P., Shields, J.A., Dua, H.S. and Donoso, L.A. (1994) Identification and rapid screening of a DraI RFLP by PCR in the retinoblastoma gene. The British journal of ophthalmology 78(2), 159.
Bookstein, R., Lai, C.-C., Hoang, T. and Lee, W.-H. (1990) PCR-based detection of a polymorphic BamHI site in intron 1 of the human retinoblastoma (RB) gene. Nucleic acids research 18(6), 1666-1666.
Valasek, M.A. and Repa, J.J. (2005) The power of real-time PCR. Advances in Physiology Education 29(3), 151-159.
Zentilin, L. and Giacca, M. (2007) Competitive PCR for precise nucleic acid quantification. Nature Protocols 2(9), 2092-2104.
Dorigo, U., Volatier, L. and Humbert, J.F. (2005) Molecular approaches to the assessment of biodiversity in aquatic microbial communities. Water Research 39(11), 2207-2218.
Zhang, T. and Fang, H.H.P. (2006) Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples. Applied Microbiology and Biotechnology 70(3), 281-289.
Johansson, M.K., Fidder, H., Dick, D. and Cook, R.M. (2002) Intramolecular dimers: A new strategy to fluorescence quenching in dual-labeled oligonucleotide probes. Journal of the American Chemical Society 124(24), 6950-6956.
Moreira, B.G., You, Y., Behlke, M.A. and Owczarzy, R. (2005) Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability. Biochemical and Biophysical Research Communications 327(2), 473-484.
Gilbride, K., Lee, D.-Y. and Beaudette, L. (2006) Molecular techniques in wastewater: understanding microbial communities, detecting pathogens, and real-time process control. Journal of microbiological methods 66(1), 1-20.
Tobler, N.E., Pfunder, M., Herzog, K., Frey, J.E. and Altwegg, M. (2006) Rapid detection and species identification of Mycobacterium spp. using real-time PCR and DNA-Microarray. Microbiological Methods 66, 116-124.
Michinaka, A., Yen, H., Chiu, Y., Tsao, H. and Lin, T. (2012) Rapid on-site multiplex assays for total and toxigenic Microcystis using real-time PCR with microwave cell disruption. Water science and technology: a journal of the International Association on Water Pollution Research 66(6), 1247.
王昱澤 (2012) 多通量定量聚合酶鏈鎖反應技術偵測水中有害藍綠菌之發展與現地應用。林財富教授。國立成功大學環境工程系碩士學位論文。
Al-Tebrineh, J., Pearson, L.A., Yasar, S.A. and Neilan, B.A. (2012) A multiplex qPCR targeting hepato-and neurotoxigenic cyanobacteria of global significance. Harmful Algae 15, 19-25.
Rasmussen, J.P., Giglio, S., Monis, P.T., Campbell, R.J. and Saint, C.P. (2008) Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria. J Appl Microbiol 104(5), 1503-1515.
Palenik, B. and Haselkorn, R. (1992) Multiple evolutionary origins of prochlorophytes, the chlorophyllb-containing prokaryotes.
Wang, L., Li, Y. and Mustapha, A. (2007) Rapid and simultaneous quantitation of Escherichia coli 0157: H7, Salmonella, and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation. Journal of Food Protection 70, 1366-1372.
Hyeon, J.-Y., Park, C., Choi, I.-S., Holt, P.S. and Seo, K.-H. (2010) 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 144(1), 177-181.
Dai, J., Peng, H., Chen, W., Cheng, J. and Wu, Y. (2013) Development of multiplex real‐time PCR for simultaneous detection of three Potyviruses in tobacco plants. Journal of applied microbiology 114(2), 502-508.