Establishment and field applications of real-time PCR methods for the quantification of potential MIB-producing cyanobacteria in aquatic systems

Abstract

2-Methylisoborneol (MIB), along with geosmin, has been frequently associated with taste and odor (T&O) events in waters, and cyanobacterial species were considered the main producers in freshwater ecosystems. The detection and quantification of genes responsible for the synthesis of these odors on-site could be valuable for the prediction and treatment of T&O. In this study, SYBR Green and TaqMan real-time quantitative polymerase chain reaction (qPCR) assays were established on the basis of conserved regions in cyanobacterial MIB cyclase gene (mic) and applied to the quantification of potential MIB-producing cyanobacteria in waters. These two qPCR assays showed the prescribed values of amplification efficiency (0.976 and 1.023, respectively) with linearity (R 2 >0.99) in the range of 10 1 to 10 7 mic copies. Examination on environmental samples and cyanobacterial cultures indicated that qPCRs were rarely inhibited by background biomass, and the limit of quantification was at the level of 10 3 mic copies L −1 in natural waters. The seasonal variations of mic abundances in Lushui Reservoir, Donghu Lake, and Qinghai Lake of China were successfully quantified using the two qPCR protocols and indicated the applicability of these assays in field monitoring. Regression analysis revealed the positive correlation between mic abundance and MIB concentration, but no significant linear correlation was found between MIB-producing cyanobacteria and total cyanobacteria in investigated waters. The qPCR assays developed in this study could rapidly and reliably detect and quantify the abundance of potential cyanobacterial MIB producers, thereby providing a valuable tool for the prediction and early detection of T&O events.

Biochemistry and genetics of taste- and odor-producing cyanobacteria

“…Most notably, Su et al (2015) and Wang et al (2015b) both report defined relationships between cells/L, gene copies/L, and 2-MIB concentration/L. Working with Pseudanabaena sp.…”

“…Working with Pseudanabaena sp. dqh15, Wang et al (2015b) estimated threshold levels of approximately 10 5 / cells/L and gene copies/L for a ''detectable'' 2-MIB odor, based on the assumption of a single MIB operon per cyanobacterial genome, which may not hold for other cyanobacteria. Both studies estimate 2-MIB/cell or 2-MIB/gene to be $10-60 fg in both culture and field samples, and it is the reasonable linear correlation that has provided users with the potential to use these data as part of a risk management approach.…”

Elucidation of Taste- and Odor-Producing Bacteria and Toxigenic Cyanobacteria in a Midwestern Drinking Water Supply Reservoir by Shotgun Metagenomic Analysis

“…Each qPCR assay utilized a positive-control synthetic gene standard (gBlock; Integrated DNA Technologies) that consisted of double-stranded DNA identical to the genes of interest identified from the metagenomes (see Table S2 in the supplemental material). The gBlock was used as the positive control for all taste-and-odor PCR analyses and contained three different amplicon targets corresponding to an Anabaena geosmin synthase (geoA) identified in the present study, a heterotrophic bacterium-specific geosmin synthase (29) and a universal cyanobacterial MIB synthase (30). A second gBlock contained part of a Microcystis-specific microcystin synthetase E (mcyE) gene that has been adapted for TaqMan qPCR (see Table S2) (31,32).…”

“…MIB synthase were revealed. The recruited sequencing reads indicated that recently described cyanobacterial MIB primers (13,30) should be able to amplify this MIB synthase from Cheney Reservoir cyanobacteria (see Table S2 in the supplemental material). Endpoint PCR incorporating both primer sets was applied to all study samples, with only two samples amplifying, corresponding to the 9 September and 25 September 2013 dates.…”

Molecular Probes to Evaluate the Synthesis and Production Potential of an Odorous Compound (2-methylisoborneol) in Cyanobacteria

“…In particular, because even rarely occurring species of cyanobacteria in certain freshwater systems can produce odorous substances, it is necessary to apply highly sensitive and specific methods. Molecular biological approaches such as polymerase chain reaction (PCR), are very relevant in this regard [13,14]. The biochemical mechanisms and genes involved in 2-MIB synthesis in various cyanobacteria genera, including Pseudanabaena, Planktothricoides, Lyngbya, and Oscillatoria (Limnothrix), have been revealed; therefore, cyanobacterial strains capable of synthesizing 2-MIB can now be detected by PCR analysis using sequence-specific primers for the mibC gene encoding a monoterpene cyclase, which is the main enzyme involved in 2-MIB synthesis [12,[15][16][17][18].…”