Abstract
Freshwater algal blooms have become a growing concern all around the world, which are caused by a high level of cyanobacteria, particularly Microcystis spp. and C. raciborskii that can produce microcystin and cylindrospermopsin, respectively. Long-time exposure to these cyanotoxins may affect public health; thus, reliable detection and quantification of the algae species are challenging in water quality management. The traditional manual counting mainly involves microscopic identification and counting of cells, which are limited by inaccuracy and time-consuming. With the development of molecular techniques and an increasing number of microbial sequences available in the GenBank database, the use of molecular methods can be used for more rapid, reliable, and accurate detection and quantification. In this study, polymerase chain reaction (PCR), multiplex PCR, and real-time quantitative PCR (qPCR) techniques were developed and applied for monitoring cyanobacteria Microcystis spp. and C. raciborskii in Macau Storage Reservoir. The results showed that the techniques were successful for identifying and quantifying the species in pure cultures and mixed cultures and were proved to be potential application to water samples in MSR. When the target species were above 1 million cells/L, similar cell numbers estimated by microscopic counting and qPCR were obtained. Further quantification in water samples indicated that the ratio of cell number estimated by microscopy and by qPCR was 0.4:12.9 for cyanobacteria and 0.2:3.9 for C. raciborskii. However, Microcystis spp. was not observed by manual counting, while it can be detected at low levels by qPCR, suggesting that qPCR is proved to be more sensitive and accurate than microscopic counting. Thus the molecular approaches provide another reliable monitoring option, in addition to the traditional microscopic counting, for ecosystem monitoring program.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Alster A, Kaplan-Levy RN, Sukenik A, et al. Morphology and phylogeny of a non-toxic invasive Cylindrospermopsis raciborskii from a Mediterranean Lake. Hydrobiologia. 2010;639:115–28.
Al-Tebrineh J, Pearson LA, Yasar SA, et al. A multiplex qPCR targeting hepato- and neurotoxigenic cyanobacteria of global significance. Harmful Algae. 2012;15:19–25.
Artz RRE, Avery LM, Jones DL, et al. Potential pitfalls in the quantitative molecular detection of Escherichia coli O157:H7 in environmental matrices. Can J Microbiol. 2006;52(5):482–8.
Falconer I, Bartram J, Chorus I, et al. Safe levels and safe practices. In: Chorus I, Bartram J, editors. Toxic Cyanobacteria in water, a guide to their public health consequences. London: Monitoring and Management. Spon Press; 1999. p. 161–82.
Felske A, Osborn AM. DNA fingerprinting of microbial communities. In: Osborn AM, Smith CJ, editors. Molecular microbial ecology. New York: Taylor & Francis; 2005. p. 65–90.
Giglio S, Monis PT, Saint CP. Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR. Nucleic Acids Res. 2003;31(22):136.
Ha JH, Hidaka T, Tsuno H. Quantification of toxic Microcystis and evaluation of its dominance ratio in blooms using real-time PCR. Environ Sci Technol. 2009;43(3):812–8.
Hawkins PR, Chandrasena NR, Jones GJ, et al. Isolation and toxicity of Cylindrospermopsis raciborskii from an ornamental lake. Toxicon. 1997;35(3):314–46.
Humbert JF, Quiblier C, Gugger M. Molecular approaches for monitoring potentially toxic marine and freshwater phytoplankton species. Anal Bioanal Chem. 2010;397(5):1723–32.
Kurmayer R, Kutzenberger T. Application of real-time PCR for quantification of microcystin genotypes in a population of the toxic cyanobacterium Microcystis sp. Appl Environ Microb. 2003;69(11):6723–30.
Larionov A, Krause A, Miller W. A standard curve based method for relative real time PCR data processing. BMC Bioinform. 2005;6:62.
McAlice BJ. Phytoplankton sampling with the Sedgewick Rafter Cell. Limnol Oceanogr. 1971;16(1):19–28.
Moreira C, Martins A, Azevedo J, et al. Application of real-time PCR in the assessment of the toxic cyanobacterium Cylindrospermopsis raciborskii abundance and toxicological potential. Appl Microbiol Biot. 2011;92(1):189–97.
Nubel U, Garcia-Pichel F, Muyzer G. PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl Environ Microb. 1997;63(8):3327–32.
Oh KH, Jeong DH, Shin SH, et al. Simultaneous quantification of cyanobacteria and Microcystis spp. using Real-Time PCR. J Microbiol Biotechnol. 2012;22(2):248–55.
Ohtani I, Moore RE, Runnegar MTC. Cylindrospermopsin: a potent hepatotoxin from the blue-green alga Cylindrospermopsis raciborskii. J Am Chem Soc. 1992;114(20):7941–2.
Otsuka S, Suda S, Li R, et al. 16S rDNA sequences and phylogenetic analyses of Microcystis strains with and without phycoerythrin. FEMS Microbiol Lett. 1998;164(1):119–24.
Paerl HW, Huisman J. Climate change: a catalyst for global expansion of harmful cyanobacterial blooms. Environ Microbiol Rep. 2009;1(1):27–37.
Pearson LA, Neilan BA. The molecular genetics of cyanobacterial toxicity as a basis for monitoring water quality and public health risk. Curr Opin Biotechnol. 2008;19(3):281–8.
Philip OT, Rasmussen JP, Michele BA, et al. Evaluation of quantitative real-time PCR to characterise spatial and temporal variations in cyanobacteria, Cylindrospermopsis raciborskii (Woloszynska) Seenaya et Subba Raju and cylindrospermopsin concentrations in three subtropical Australian reservoirs. Harmful Algae. 2010;9(3):243–54.
Pidasak J. Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju, an expanding highly adaptive cyanobacterium: worldwide distribution and review of its ecology. Arch Hydrobiol Suppl. 1997;107:563–93.
Rasmussen JP, Giglio S, Monis PT, et al. Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria. J Appl Microbiol. 2008;104(5):1503–15.
Rinta-Kanto JM, Ouellette AJA, Boyer GL, et al. Quantification of toxic Microcystis spp. during the 2003 and 2004 blooms in Western Lake Erie using quantitative real-time PCR. Environ Sci Technol. 2005;39(11):4198–205.
Scholin CA, Herzog M, Sogin M, et al. Identification of group- and strain-specific genetic markers for globally distributed Alexandrium (Dinophyceae) species II. Sequence analysis of a fragment of the large-subunit ribosomal RNA gene. J Phycol. 1994;30:999–1011.
Schoske R, Vallone PM, Ruitberg CM, et al. Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci. Anal Bioanal Chem. 2003;375(3):333–43.
Tomioka N, Nagai T, Kawasaki T, et al. Quantification of Microcystis in a Eutrophic Lake by simple DNA extraction and SYBR Green real-time PCR. Microbes Environ. 2008;23(4):306–12.
Vaitomaa J, Rantala A, Halinen K, et al. Quantitative real-time PCR for determination of Microcystin synthetase E copy numbers for Microcystis and Anabaena in Lakes. Appl Microbiol Biotechnol. 2003;69(12):7289–97.
WHO. Guidelines for drinking-water quality. Second edition, Addendum to vol 2, Health criteria and other supporting information. Geneva: World Health Organisation; 1998.
Wilson KM, Schembri MA, Baker PD, et al. Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR. Appl Environ Microbiol. 2000;66(1):332–8.
Wittwer CT, Herrmann MG, Moss AA, et al. Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques. 1997;22(1):130–8.
Zhang W, Lou I, Ung WK, et al. Eutrophication analyses and principle component regression for two subtropical storage reservoirs in Macau. Desalin Water Treat. 2013;51(37–39):7331–40.
Zhang W, Lou I, Ung WK, et al. Spatio-temporal variations of phytoplankton structure and water quality in the eutrophic freshwater reservoir of Macau. Desalin Water Treat. 2014;55(8):1–16.
Acknowledgments
We thank Larrisa Lei, the master student in the Institute of Chinese Medical Science, for assistance with PCR and gel electrophoresis studies, the technical staffs at Macao Water Supply Co. Ltd. for water sampling, and Xi Chen, the undergraduate student in the Department of Civil and Environmental Engineering at University of Macau, for helping in DNA extraction and water parameter measurement.
The research project was supported by the Fundo para o Desenvolvimento das Ciências e da Tecnologia (FDCT), under grant No. 016/2011/A, and the Research Committee of University of Macau, under grant No. MRG002/LIC/2012/FST.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media Dordrecht
About this chapter
Cite this chapter
Zhang, W., Lou, I., Ung, W.K., Kong, Y., Mok, K.M. (2017). Application of PCR and Real-Time PCR for Monitoring Cyanobacteria, Microcystis spp. and Cylindrospermopsis raciborskii, in Macau Freshwater Reservoir. In: Lou, I., Han, B., Zhang, W. (eds) Advances in Monitoring and Modelling Algal Blooms in Freshwater Reservoirs. Springer, Dordrecht. https://doi.org/10.1007/978-94-024-0933-8_5
Download citation
DOI: https://doi.org/10.1007/978-94-024-0933-8_5
Published:
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-024-0931-4
Online ISBN: 978-94-024-0933-8
eBook Packages: Earth and Environmental ScienceEarth and Environmental Science (R0)