USGS Ohio Water Microbiology Laboratory - Cyanobacteria and toxin gene molecular assays

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Harmful cyanobacterial “algal” blooms and associated toxins are of concern in many parts of the world because of their effects on drinking water, water-based recreation, and watershed ecology. Cyanotoxins are a diverse group of compounds that include hepatotoxins, neurotoxins, cytotoxins, dermatotoxins, and irritant toxins (Wiegand and Pflugmacher, 2005). Numerous incidents of animal and human poisonings associated with toxic cyanobacterial blooms have been reported (Bláha and others, 2009).

USGS Ohio Water Microbiology Laboratory - Cyanobacteria and toxin gene molecular assays
USGS Ohio Water Microbiology Laboratory - Cyanobacteria and toxin gene molecular assays.

Not all cyanobacterial genera have the ability to produce toxins and some genera have the ability to produce more than one type of toxin. For toxin production to occur, a gene coding for a specific toxin must be present in the genome of the cyanobacterial cell. Known toxin-producing genera include both toxic strains (with the toxin gene) and nontoxic strains (without the toxin gene), which can be differentiated only by molecular detection methods such as quantitative polymerase chain reaction (qPCR). 

To better understand and predict cyanobacterial toxin production, the Ohio Water Microbiology Laboratory (OWML) developed the capability to analyze samples by several qPCR assays.   By using qPCR, a targeted genetic sequence of DNA or RNA is amplified into an amount that can be quantified.  Whereas DNA-based qPCR methods reveal the presence of toxin genes (irrespective of whether they are actively producing toxin), RNA-based methods can detect toxin-producing cyanobacteria that are actively expressing the toxin genes (Sipari et al., 2010).  The USGS Ohio Water Microbiology Laboratory (OWML) has the capability to run qPCR assays on four levels:  

  1. General cyanobacteria
  2. General assays for the genera Microcystis, Planktothrix, and Anabaena/Dolichospermum
  3. DNA-based toxin gene assays for microcystin, saxitoxin, anatoxin-a, and cylindrospermopsin
  4. RNA-based toxin gene assays for all toxins listed in the third level.  

General cyanobacteria are detected as described in Rinta-Kanto and others (2005). This DNA-based assay targets general cyanobacterial genes using the primers CYAN108F and CYAN377R and probe CYAN328P.   The assay developers confirmed that this assay can detect different cyanobacterial cultures including Microcystis, Planktothrix, Anabaena, Synechococcus, and Synechocystis.     

General  Microcystis are detected as described in Rinta-Kanto and others (2005).  This DNA-based assay targets genes present in the genus Microsystis using the primers MICR184F and MICR431R and probe MICR228P.  This assay detects both toxic and non-toxic strains of Microcystis

General  Planktothrix are detected as described in Ostermaier and Kurmayer (2009).  This DNA-based assay targets genes present in the genus Planktothrix using the primers PlankF and Plank R and probe PlankP.  This assay detects both toxic and non-toxic strains of Planktothrix.

General  Anabaena/Dolichospermum are detected as described in Doblin and others (2007).  This DNA-based assay targets genes present in the genus Anabaena/Dolichospermum using the primers Ana573F and Ana780R and a SYBR® Green probe (Applied Biosystems, Carlsbad, Cal.).  This assay detects both toxic and non-toxic strains of Anabaena/Dolichospermum.

The following assays can be used to identify the frequency of occurrence and compositions of potential toxin-producing cyanobacteria. 

Universal microcystin toxin genes (mcyE) are detected as described in Rantala and others (2004) and can be used for both DNA and RNA detection. This assay targets a portion of the mcyE toxin gene using the primers mcyE-F2 and mcyE-R4 and a SYBR® Green probe (Applied Biosystems, Carlsbad, Cal.).  This assay targets the mcyE toxin gene in all major microcystin-producing genera.

Microsystis-, Anabaena-, and Planktothrix genera- specific microcystin toxin genes (mcyE) can be used for both DNA and RNA detection.  These assays target the following primers and probes: 

  • Microcystis-specific  mcyE primers 127F, 247R, and probe 186P (Sipari and others, 2010)
  • Planktothrix-specific mcyE primers mcyE-F2, mcyE-plaR3, and SYBR® Green probe (Applied Biosystems, Carlsbad, Cal.) (Vaitomaa and others, 2003; Rantala and others, 2006).
  • Anabaena-specific mcyE primers 611F, 737R, and probe 672P (Sipari and others, 2010)

Universal saxitoxin toxin genes (sxtA) are detected as described in Al-Tebrineh and others (2012) and can be used for both DNA and RNA detection. This assay targets a portion of the sxtA toxin gene using the primers sxtF and sxtR and probe sxtP. This assay targets the sxtA gene in all major saxitoxin-producing cyanobacteria.

Universal anatoxin-a toxin genes (anaC) are detected as described in Sabart and others (2015) and can be used for both DNA and RNA detection. This assay targets a portion of the anaC toxin gene using the primers anaC-gen-F2 and anaC-gen-R2 and a SYBR® Green probe (Applied Biosystems, Carlsbad, Cal.). This assay targets the anaC gene in all major anatoxin-a-producing cyanobacteria.

Universal cylindrospermopsin toxin genes (cyrA) are detected as described in Al-Tebrineh and others (2012) and can be used for both DNA and RNA detection. This assay targets a portion of the cyrA toxin gene using the primers cyrF and cyrR and probe cyrP.  This assay targets the cyrA gene in all major cylindrospermopsin- producing cyanobacteria.

The OWML will continue to test and apply new assays for cyanobacteria and cyanobacterial toxin genes as needed for future projects.

References

Al-Tebrineh, J., Pearson, L.A., Yasar, S.A., Neilan, B.A., 2012, A multiplex qPCR targeting hepato- and neurotoxigenic cyanobacteria of global significance: Harmful Algae, v. 15, p. 19-25.

Bláha, L., Babica, P., Maršálek, B., 2009, Toxins produced in cyanobacterial water blooms – toxicity and risks: Interdiscip Toxicol, v. 2., p. 36-41.

Doblin, M.A., Coyne, K.J., Rinta-Kanto, J.M., Wilhelm, S.W., Dobbs, F.C., 2007, Dynamics and short-term survival of toxic cyanobacteria species in ballast water from NOBOB vessels transiting the Great lakes—implications for HAB invasions: Harmful Algae, v. 6., p. 519-530.

Ostermaier, V., Kurmayer, R., 2009, Distribution and abundance of nontoxic mutants of cyanobacteria in lakes of the Alps: Microbiology of Aquatic Systems, v. 58, p. 323-333.

Rantala, A., Fewer, D.P., Hisbergues, M., Rouhiainen, L., Vaitomaa, J., Borner, T., Sivonen, K., 2004, Phylogenetic evidence for the early evolution of microcystin synthesis:  Proceedings of the National Academy of Science, v. 101, no. 2, p. 568-573.

Rantala, A., Rajaniemi-Wacklin, P., Lyra, C., Lepisto, L., Rintala, J., Mankiewicz-Boczek, J., 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: Applied and Environmental Microbiology, v. 72, p. 6101-6110.

Rinta-Kanto, J.M., Ouellette, A.J.A., Boyer, G.L., Twiss, M.R., Bridgeman, T.B., Wilhelm, S.W.,  2005, Quantification of toxic Microcystis spp. during the 2003 and 2004 blooms in western Lake Erie using quantitative real-time PCR:  Environmental Science and Technology, v. 39, p. 4198-4205.

Sabart, M., Crenn, K., Perriere, F., Abila, A., Leremboure, M., Colombet, J., Jousse, C., Latour, D., 2015, Co-occurance of microcystin and anatoxin-a in the freshwater lake Aydat (France): analytical and molecular approaches during a three-year survey: Harmful Algae, v. 48, p. 12-20.

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:  Applied and Environmental Microbiology, v. 76, p. 3797-3805.

Sivonen, K.,  2008, Emerging high throughput analyses of cyanobacterial toxins and toxic cyanobacteria: In H. K. Hudnell (ed.), Advances in experimental medicine and biology, Cyanobacterial harmful algal blooms—state of the science and research needs, Springer, New York, NY, v. 619, p. 539-557.

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: Applied and Environmental Microbiology, v. 69, no. 12, p. 7289–7297.

Wiegand, C., Pflugmacher, S., 2005, Ecotoxicological effects of selected cyanobacterial secondary metabolites a short review: Toxicology and Applied Pharmacology, v. 203, p. 201-218.