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.