Relations between DNA- and RNA-based molecular methods for cyanobacteria and microcystin concentration at Maumee Bay State Park Lakeside Beach, Oregon, Ohio, 2012

Abstract

Water samples were collected from Maumee Bay State Park Lakeside Beach, Oregon, Ohio, during the 2012 recreational season and analyzed for selected cyanobacteria gene sequences by DNA-based quantitative polymerase chain reaction (qPCR) and RNA-based quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results from the four DNA assays (for quantifying total cyanobacteria, total Microcystis, and Microcystis and Planktothrix strains that possess the microcystin synthetase E (mcyE) gene) and two RNA assays (for quantifying Microcystis and Planktothrix genera that are expressing the microcystin synthetase E (mcyE) gene) were compared to microcystin concentration results determined by an enzyme-linked immunosorbent assay (ELISA). Concentrations of the target in replicate analyses were log 10 transformed. The average value of differences in log 10 concentrations for the replicates that had at least one detection were found to range from 0.05 to >0.37 copy per 100 milliliters (copy/100 mL) for DNA-based methods and from >0.04 to >0.17 copy/100 mL for RNA-based methods. RNA has a shorter half-life than DNA; consequently, a 24-hour holding-time study was done to determine the effects of holding time on RNA concentrations. Holding-time comparisons for the RNA-based Microcystis toxin mcyE assay showed reductions in the number of copies per 100 milliliters over 24 hours. The log difference between time 2 hours and time 24 hours was >0.37 copy/100 mL, which was higher than the analytical variability (log difference of >0.17 copy/100 mL). Spearman's correlation analysis indicated that microcystin toxin concentrations were moderately to highly related to DNA-based assay results for total cyanobacteria (rho=0.69), total Microcystis (rho=0.74), and Microcystis strains that possess the mcyE gene (rho=0.81). Microcystin toxin concentrations were strongly related with RNA-based assay results for Microcystis mcyE gene expression (rho=0.95). Correlation analysis could not be done for Planktothrix mcyE gene expression because of too few detections.

Cyanotoxin occurrence in large rivers of the United States

“…Primer and probe information, as well as run conditions, can be found in each of the above cited references. Details of sample concentration, extraction, and qPCR methods are described in Stelzer et al (2013). Plasmid standards for each assay were used to establish standard curves for quantification.…”

Predicting microcystin concentration action-level exceedances resulting from cyanobacterial blooms in selected lake sites in Ohio

“…Molecular assays for cyanobacteria associated with microcystin production were done to enumerate (1) general cyanobacteria (16S rRNA); (2) general Microcystis, Dolichospermum, and Planktothrix (16S rRNA); and (3) microcystin toxin genes (mcyE) for Microcystis, Dolichospermum, and Planktothrix (Doblin et al 2007;Ostermaier and Kurmayer 2009;Rantala et al 2006;Rinta-Kanto et al 2005;Sipari et al 2010;Vaitomaa et al 2003). DNA extraction/purification, standard curve, and limit of detection/quantification calculation procedures are presented elsewhere (Francy et al 2015); sample inhibition was determined according to procedures in Stelzer et al (2013). Standard curve and limits of detection and quantification data are listed for the current study (Table S1 in supplemental materials).…”

Recruitment of cyanobacteria by reverse transcription quantitative real-time PCR based on expression of Microcystis gene

“…However, qRT-PCR is known to be more sensitive and requires fewer less volume of sample RNAs and provides sequence information compared to those technologies mentioned. There are abundant studies on the quantification of synthetase genes in microcystic toxins at the DNA level, but only few have discussed these at the RNA level (Stelzer, Loftin & Struffolino, 2013). Hence, recovering rRNA sequences from RNA templates using RT-PCR implies that the biological source is active at sampling and is a good indicator of active cells.…”