Journal of Microbiology and Biotechnology
The Korean Society for Microbiology and Biotechnology publishes the Journal of Microbiology and Biotechnology.

2017 ; Vol.27-4: 808~815

AuthorEun-Hee Lee, Kyung-Suk Cho, Ahjeong Son
Place of dutyDepartment of Environmental Science and Engineering, Ewha Womans University, Seoul 03760, Republic of Korea
TitleDetection and Quantification of Toxin-Producing Microcystis aeruginosa Strain in Water by NanoGene Assay
PublicationInfo J. Microbiol. Biotechnol.2017 ; Vol.27-4
AbstractWe demonstrated the quantitative detection of a toxin-producing Microcystis aeruginosa (M. aeruginosa) strain with the laboratory protocol of the NanoGene assay. The NanoGene assay was selected because its laboratory protocol is in the process of being transplanted into a portable system. The mcyD gene of M. aeruginosa was targeted and, as expected, its corresponding fluorescence signal was linearly proportional to the mcyD gene copy number. The sensitivity of the NanoGene assay for this purpose was validated using both dsDNA mcyD gene amplicons and genomic DNAs (gDNA). The limit of detection was determined to be 38 mcyD gene copies per reaction and 9 algal cells/ml water. The specificity of the assay was also demonstrated by the addition of gDNA extracted from environmental algae into the hybridization reaction. Detection of M. aeruginosa was performed in the environmental samples with environmentally relevant sensitivity (~105 algal cells/ml) and specificity. As expected, M. aeruginosa were not detected in nonspecific environmental algal gDNA over the range of 2 × 100 to 2 × 107 algal cells/ml.
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Key_wordAlgal bloom, cyanobacteria, Microcystis aeruginosa, microcystin, NanoGene assay
References
  1. Esposito A. 2016. Chile’s salmon farms losing up to $800 million from algal bloom. Reuters, USA.
  2. Hallegraeff GM. 2010. On the global increase of harmful algal bloom. Wetl. Aust. J. 12: 2-15.
  3. Johns DG, Reid PC. 2001. An overview of plankton ecology in the North Sea, United States. Technical report produced for Strategic Environmental Assessment. SAHFOS, Plymouth, UK.
  4. Foster JM. 2013. Lake Erie is dying again, and warmer waters and wetter weather are to blame. Available at https://thinkprogress.org/lake-erie-is-dying-again-and-warmer-watersand-wetter-weather-are-to-blame-96956c15f046#.6pe6d0i0c.
  5. Kotak BG, Zurawell RW, Prepas EE, Holmes CF. 1996. Microcystin-LR concentration in aquatic food web compartments from lakes of varying trophic status. Can. J. Fish. Aquat. Sci. 53: 1974-1985.
    CrossRef
  6. Xue Q, Su X, Steinman AD, Cai Y, Zhao Y, Xie L. 2016. Accumulation of microcystins in a dominant chironomid larvae (Tanypus chinensis) of a large, shallow and eutrophic Chinese lake, Lake Taihu. Sci. Rep. 6: 31097.
    Pubmed CrossRef Pubmed Central
  7. Campos A, Vasconcelos V. 2010. Molecular mechanisms of microcystin toxicity in animal cells. Int. J. Mol. Sci. 11: 268-287.
    Pubmed CrossRef Pubmed Central
  8. Jochimsen EM, Carmichael WW, An JS, Cardo DM, Cookson ST, Holmes CEM, et al. 1998. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N. Engl. J. Med. 338: 873-878.
    Pubmed CrossRef
  9. Sivonen K, Jones G. 1999. Cyanobacterial toxins, pp. 41-111. In Chorus I, Bartram J (eds.). Toxin Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring, and Management. E & FN Spon, London, UK, for World Health Organization, Routledge.
  10. Fleming LE, Rivero C, Burns J, Williams C, Bean JA, Shea KA, Stinn J. 2002. Blue green algal (cyanobacterial) toxins, surface drinking water, and liver cancer in Florida. Harmful Algae 1: 157-168.
    CrossRef
  11. Paerl H. 2008. Nutrient and other environmental controls of harmful cyanobacterial blooms along the freshwater–marine continuum, pp. 217-237. In Hudnell HK (ed.). Cyanobacterial Harmful Algal Blooms: State of the Science and Research Needs. Springer, New York. USA.
    Pubmed CrossRef
  12. Dittmann E, Fewer DP, Neilan BA. 2013. Cyanobacterial toxins: biosynthetic routes and evolutionary roots. FEMS Microbiol. Rev. 37: 23-43.
    Pubmed CrossRef
  13. Rinta-Kanto JM, Ouellette AJ, Boyer GL, Twiss MR, Bridgeman TB, W ilhelm SW. 2 005. Q uantification o f toxic Microcystis spp. during the 2003 and 2004 blooms in western Lake Erie using quantitative real-time PCR. Environ. Sci. Technol. 39: 4198-4205.
  14. Vaitomaa J, Rantala A, Halinen K, Rouhiainen L, Tallberg P, Mokelke L, Sivonen K. 2003. Quantitative real-time PCR for determination of microcystin synthetase E copy numbers for Microcystis and Anabaena in lakes. Appl. Environ. Microbiol. 69: 7289-7297.
    Pubmed CrossRef Pubmed Central
  15. Furukawa K, Noda N, Tsuneda S, Saito T, Itayama T, Inamori Y. 2006. Highly sensitive real-time PCR assay for quantification of toxic cyanobacteria based on microcystin synthetase A gene. J. Biosci. Bioeng. 102: 90-96.
    Pubmed CrossRef
  16. Johnson BN, Mutharasan R. 2013. A cantilever biosensor-based assay for toxin-producing cyanobacteria Microcystis aeruginosa using 16S rRNA. Environ. Sci. Technol. 47: 12333-12341.
    Pubmed CrossRef
  17. Rudi K, Skulberg OM, Larsen F, Jakobsen KS. 1998. Quantification of toxic cyanobacteria in water by use of competitive PCR followed by sequence-specific labeling of oligonucleotide probes. Appl. Environ. Microbiol. 64: 2639-2643.
    Pubmed Pubmed Central
  18. Sipari H, Rantala-Ylinen A, Jokela J, Oksanen I, Sivonen K. 2010. Development of a chip assay and quantitative PCR for detecting microcystin synthetase E gene expression. Appl. Environ. Microbiol. 76: 3797-3805.
    Pubmed CrossRef Pubmed Central
  19. Lee E-H, Lim H J, S on A , Chua B . 2 015. A disposable bacterial lysis cartridge (BLC) suitable for an in situ waterborne pathogen detection system. Analyst 140: 7776-7783.
    Pubmed CrossRef
  20. Lee E-H, C hua B, S on A . 2015. M icro c orona discharge based cell lysis method suitable for inhibitor resistant bacterial sensing systems. Sensor. Actuat. B Chem. 216: 17-23.
  21. Mitchell KA, Chua B, Son A. 2014. Development of first generation in-situ pathogen detection system (Gen1-IPDS) based on NanoGene assay for near real time E. coli O157:H7 detection. Biosens. Bioelectron. 54: 229-236.
    Pubmed CrossRef
  22. Kim GY, Wang XF, Ahn H, Son A. 2011. Gene quantification by the NanoGene assay is resistant to inhibition by humic acids. Environ. Sci. Technol. 45: 8873-8880.
    Pubmed CrossRef
  23. Wang XF, Liles MR, Son A. 2013. Quantification of Escherichia coli O157:H7 in soils using an inhibitor-resistant NanoGene assay. Soil Biol. Biochem. 58: 9-15.
    CrossRef
  24. Kim GY, Son A. 2010. Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification. Anal. Chim. Acta 677: 90-96.
    Pubmed CrossRef
  25. Ouellette AJA, Wilhelm SW. 2003. Toxic cyanobacteria: the evolving molecular toolbox. Front. Ecol. Environ. 1: 359-366.
    CrossRef
  26. Kaebernick M, Neilan BA, Borner T, Dittmann E. 2000. Light and the transcriptional response of the microcystin biosynthesis gene cluster. Appl. Environ. Microbiol. 66: 3387-3392.
    Pubmed CrossRef Pubmed Central
  27. Zuker M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31: 3406-3415.
    Pubmed CrossRef Pubmed Central
  28. Joung SH, Oh HM, Ko SR, Ahn CY. 2011. Correlations between environmental factors and toxic and non-toxic Microcystis dynamics during bloom in Daechung Reservoir, Korea. Harmful Algae 10: 188-193.
    CrossRef
  29. UTEX. 2016. Medium instructions of the UTEX Culture Collection of Algae, University of Texas at Austin.
  30. Wang X, Son A. 2013. Effects of pretreatment on the denaturation and fragmentation of genomic DNA for DNA hybridization. Environ. Sci. Process. Impacts 15: 2204-2212.
    Pubmed CrossRef
  31. Padovan A. 1992. Isolation and culture of five species of freshwater algae from the alligator rivers region, Northern territory. Technical Memorandum 37. Australian Government Publishing Services, Canberra. Australia.
  32. Steel AB, L evicky RL, H erne T M, Tarlov M J. 2 000. Immobilization of nucleic acids at solid surfaces: effect of oligonucleotide length on layer assembly. Biophys. J. 79: 975981.
  33. Lim SH, Bestvater F, Buchy P, Mardy S, Yu ADC. 2009. Quantitative analysis of nucleic acid hybridization on magnetic particles and quantum dot-based probes. Sensors (Basel) 9: 5590-5599.
    Pubmed CrossRef Pubmed Central
  34. Armbruster DA, Pry T. 2008. Limit of blank, limit of detection and limit of quantitation. Clin. Biochem. Rev. 29: S49-S52.
    Pubmed Pubmed Central
  35. Blahova L, Babica P, Adamovsky O, Kohoutek J, Marsalek B, Blaha L. 2008. Analyses of cyanobacterial toxins (microcystins, cylindrospermopsin) in the reservoirs of the Czech Republic and evaluation of health risks. Environ. Chem. Lett. 6: 223-227.
    CrossRef
  36. Committee AM. 1987. Recommendations for the definition, estimation and use of the detection limit. Analyst 112: 199-204.
    CrossRef
  37. Shrivastava A, Gupta VB. 2011. Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron. Young Sci. 2: 21-25.
    CrossRef
  38. Foulds IV, Granacki A, Xiao C, Krull UJ, Castle A, Horgen PA. 2002. Quantification of microcystin-producing cyanobacteria and E. coli in water by 5’-nuclease PCR. J. Appl. Microbiol. 93: 825-834.
    Pubmed CrossRef
  39. Kim GY, Wang XF, Son A. 2011. Inhibitor resistance and in situ capability of nanoparticle based gene quantification. J. Environ. Monitor. 13: 1344-1350.
    Pubmed CrossRef
  40. Wang X, Lim HJ, Son A. 2014. Characterization of denaturation and renaturation of DNA for DNA hybridization. Environ. Health Toxicol. 29: e2014007.
    Pubmed CrossRef Pubmed Central
  41. Oberholster P, Botha A, Grobbelaar J. 2004. Microcystis aeruginosa: source of toxic microcystins in drinking water. Afr. J. Biotechnol. 3: 159-168.
    CrossRef
  42. Oh HM, Lee SJ, Jang MH, Yoon BD. 2000. Microcystin production by Microcystis aeruginosa in a phosphorus-limited chemostat. Appl. Environ. Microbiol. 66: 176-179.
    Pubmed CrossRef Pubmed Central
  43. Long BM, Jones GJ, Orr PT. 2001. Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate. Appl. Environ. Microbiol. 67: 278-283.
    Pubmed CrossRef Pubmed Central



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