Abstract
Based on sequence analyses of phycocyanin intergenic spacers (PC-IGS) from Microcystis, Anabaena, Aphanizomenon, and Planktothrix (Oscillatoria) strains, a genus-specific probe pair TF/TR was designed, and a sandwich hybridization assay was established to quantitatively detect Microcystis. Through BLAST and cyanobacterial culture tests, TF/TR was demonstrated to be specific for Microcystis. A calibration curve for the sandwich hybridization assay was established, and the lowest detected concentration was 100 cell/ml. Laboratory and field samples were analyzed with both sandwich hybridization assay and microscopy. The biotic and abiotic components of the samples were of little disturbance to the sandwich hybridization assay. The results showed no distinct difference between the two methods. In this study, a sandwich hybridization assay was established to detect Microcystis, providing an alternative to traditional microscopic, morphology-based methods.
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Al-Tebrineh, J., Mihali, T.K., Pomati, F., and Neilan, B.A. 2010. Detection of saxitoxin-producing Cyanobacteria and Anabaena circinalis in environmental water blooms by quantitative PCR. Appl. Environ. Microbiol. 76, 7836–7842
Anderson, D.M., Kulis, D.M., Keafer, B.A., Gribble, K.E., Marin, R., and Scholin, C.A. 2005. Identification and enumeration of Alexandrium spp. from the Gulf of Maine using molecular probes. Deep-Sea Res. Pt II 52, 2467–2490.
Baker, J.A., Neilan, B.A., Entsch, B., and McKay, D.B. 2001. Identification of cyanobacteria and their toxigenicity in environmental samples by rapid molecular analysis. Environ. Toxicol. 16, 472–482.
Baxa, D.V., Kurobe, T., Ger, K.A., Lehman, P.W., and Teh, S.J. 2010. Estimating the abundance of toxic Microcystis in the San Francisco Estuary using quantitative real-time PCR. Harmful Algae 9, 342–349.
Bolch, C.J.S., Blackburn, S.I., Neilan, B.A., and Grewe, P.M. 1996. Genetic characterization of strains of cyanobacteria using PCR-RFLP of the cpcBA intergenic spacer and anking regions. J. Phycol. 32, 445–451.
Bolch, C.J.S., Orr, P.T., Jones, G.J., and Blackburn, S.I. 1999. Genetic, morphological, and toxicological variation among globally distributed strains of Nodularia (cyanobacteria). J. Phycol. 35, 339–355.
Charmichael, W.W. 1996. Toxic microcystis and the environment, pp. 13–34. In Watanabe, M.F., Harada, K., Carmichael, W.W., and Fujiki, H. (eds.), Toxic Microcystis. CRC Press, USA.
Codd, G.A., Bell, S.G., Kaya, K., Ward, C.J., Beattie, K.A., and Meatcalf, J.S. 1999. Cyanobacterial toxins, exposure routes and human health. Eur. J. Phychol. 34, 405–415.
Kurmayer, R. and Kutzenberger, T. 2003. Application of real-time PCR for quantification of microcystin genotypes in a population of the toxic cyanobacterium Microcystis sp. Appl. Environ. Microbiol. 69, 6723–6730.
Li, X.Q., Yang, F., Yin, L.H., Li, Y.H., Li, X.B., and Pu, Y.P. 2009. Identification and toxigenicity of dominant algae of Taihu Lake bloom by whole-cell PCR method. 8th Natl. Postgrad. Symp. Environ. Occup. Med. Proc. 81–83.
Matsunaga, T., Nakayama, H., Okochi, M., and Takeyama, H. 2001. Fluorescent detection of cyanobacterial DNA using bacterial magnetic particles on a MAG-microarray. Biotechnol. Bioeng. 73, 400–405.
Mikulski, C.M., Park, Y.T., Jones, K.L., Lee, C.K., Lim, W.A., Lee, Y., Scholin, C.A., and Doucette, G.J. 2008. Development and field application of rRNA-targeted probes for the detection of Cochlodinium polykrikoides Margalef in Korean coastal waters using whole cell and sandwich hybridization formats. Harmful Algae 7, 348–359.
Neilan, B.A., Jacobs, D., and Goodman, A. 1995. Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus. Appl. Environ. Microbiol. 61, 3875–3883.
Neilan, B.A., Stuart, J.L., Goodman, A.E., Cox, P.T., and Hawkins, P.R. 1997. Specific amplication and restriction polymorphisms of the cyanobacterial rRNA operon spacer region. Syst. Appl. Microbiol. 20, 612–621.
Otsuka, S., Suda, S., Li, R.H., Watanabe, M., Oyaizu, H., Matsumoto, S., and Watanabe, M.M. 1999. Phylogenetic relationships between toxic and nontoxic strains of the genus Microcystis based on 16S to 23S internal transcribed spacer sequence. FEMS Microbiol. Lett. 172, 15–21.
Pan, H., Song, L.R., Liu, Y.D., and Borner, T. 2002. Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples. Arch. Microbiol. 178, 421–427.
Preston, C.M., Marin, R., Jensen, S.D., Feldman, J., Birch, J.M., Massion, E.I., DeLong, E.F., Suzuki, M., Wheeler, K., and Scholin, C.A. 2009. Near real-time, autonomous detection of marine bacterioplankton on a coastal mooring in Monterey Bay, California, using rRNA-targeted DNA probes. Environ. Microbiol. 11, 1168–1180.
Rantala, A., Rajaniemi-Wacklin, P., Lyra, C., Lepist_, L., Rintala, J., Mankiewicz-Boczek, J., and Sivonen, K. 2006. Detection of microcystin-producing cyanobacteria in Finnish lakes with genus-specific microcystin synthetase gene E (mcyE) PCR and associations with environmental factors. Appl. Environ. Microbiol. 72, 6101–6110.
Rippa, R., Deruelles, J., Waterbury, J., Herdman, M., and Stanier, R. 1979. Generic assignment, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1–61.
Rudi, K., Skulberg, O.M., Skulberg, R., and Jakobsen, K.S. 2000. Application of sequence-specific labeled 16S rRNA gene oligonucleotide probes for genetic profiling of cyanobacterial abundance and diversity by array hybridization. Appl. Environ. Microbiol. 66, 4004–4011.
Scholin, C.A., Buck, K.R., Britschgi, T., Cangelosi, G., and Chavez, F.P. 1996. Identification of Pseudo-nitzschia australis (Bacillariophyceae) using rRNA-targeted probes in whole cell and sandwich hybridization formats. Phycology 35, 190–197.
Semary, E.l. and Adel, N. 2010. Investigating factors affecting growth and cellular mcyB transcripts of Microcystis aeruginosa PCC 7806 using real-time PCR. Ann. Microbiol. 60, 181–188.
Tan, W., Liu, Y., Wua, Z., Lin, S., Yu, G., Yu, B., and Li, R. 2010. cpcBA-IGS as an effective marker to characterize Microcystis wesenbergii (Koma’ rek) Koma’ rek in Kondrateva (cyanobacteria). Harmful Algae 9, 607–612.
Tillett, D., Parker, D.L., and Neilan, B.A. 2001. Detection of toxigenicity by a probe for the microcystin synthetase A gene (mcyA) of the cyanobacterial genus Microcystis: comparison of toxicities with 16S rRNA and phycocyanin operon (phycocyanin intergenic spacer) phylogenies. Appl. Environ. Microbiol. 67, 2810–2818.
Tomioka, N., Nagai, T., Kawasaki, T., Imai, A., Matsushige, K., and Kohata, K. 2008. Quantification of Microcystis in a eutrophic Lake by simple DNA extraction and SYBR green real-time PCR. Microbes Environ. 23, 306–312.
Tyrrell, J.V., Scholin, C.A., Berguist, P.R., and Berguist, P.L. 2001. Detection and enumeration of Heterosigma akashiwo and Fibrocapsa japonica (Raphidophyceae) using rRNA-targeted oligonucleotide probes. Phycology 40, 457–467.
Vaitomaa, J., Rantala, A., Halinen, K., Rouhianen, L., Tallberg, P., Mokelke, L., and 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.
Zhen, Y., Mi, T., and Yu, Z. 2009. Detection of several harmful algal species by sandwich hybridization integrated with a nuclease protection assay. Harmful Algae 8, 651–657.
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Zhu, J.P., Li, X. & Du, S. Identification and enumeration of Microcystis using a sandwich hybridization assay. J Microbiol. 50, 186–190 (2012). https://doi.org/10.1007/s12275-012-1418-9
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DOI: https://doi.org/10.1007/s12275-012-1418-9