Rapid on-site multiplex assays for total and toxigenic Microcystis using real-time PCR with microwave cell disruption

Abstract

A quantitative real-time polymerase chain reaction (qPCR) is a robust means by which to monitor toxin-producing cyanobacteria. However, qPCR usually requires DNA extraction, which is a time-consuming, labor-intensive pretreatment. To be able to quickly determine the potential of cyanotoxin contamination in the field, a rapid pretreatment method for DNA extraction and a portable qPCR device are needed. In this study, we applied a microwave-based method for the qPCR pretreatment and a multicolor portable qPCR with UPL and TaqMan probes to quantify toxigenic and total Microcystis. The method was tested using laboratory cultures of toxigenic Microcystis aeruginosa PCC7820. The qPCR results showed the cycle thresholds value (Ct value) correlated well with cell numbers, with detection limit at about 1,000 cells/ml. This scheme was applied in 22 environmental samples from six drinking water reservoirs (DWRs) in Taiwan. Although the results for qPCR were about four times higher than those of microscopic observation, good correlation between qPCR and microscope methods were obtained (r-square: 0.79, P < 0.01). The ratios of toxigenic Microcystis to total Microcystis in two reservoirs, Sin-Shan Reservoir and Shih-men Reservoir, were less than 10%. In three other reservoirs, Ren-Yi-Tan Reservoir, Nan-Hua Reservoir and Bao-Shan Reservoir, much higher (>46.1%) ratios were obtained. The scheme may assist quick assessment of the risk associated with toxic cyanobacteria in DWRs.

A qPCR-Based Tool to Diagnose the Presence of Harmful Cyanobacteria and Cyanotoxins in Drinking Water Sources

“…In the last two decades, quantitative real-time polymerase chain reaction (qPCR)-based molecular techniques [4,9,45,46,47] and the enzyme-linked immuno-sorbent assay (ELISA) method [48,49] have been successfully developed and applied to detect toxigenic genes and cyanotoxins, respectively. Both of these techniques have quick turn-around times and are capable of multiple sample analyses in a single run.…”

“…Therefore, multiplex-qPCR, capable of amplifying several different targets in a single reaction well, may provide a simple approach to simultaneously detect multiple target genes. Although these methods have been well documented, most studies are limited to the monitoring of one reservoir/lake and with limited sample size [5,9,10,11,45,46,47,50,52,53,54,55,56,57]. In applying multiplex-qPCR systems, inter-influence of gene abundance on the detection has been reported by only four studies [51,58,59,60], in which less abundant genes were not detected due to the ample presence of other targeted genes.…”

Recent trends in the detection of freshwater cyanotoxins with a critical note on their occurrence in Asia

“…The portable qPCR methods were validated using laboratory toxigenic strain M. aeruginosa PCC7820. Six drinking water reservoir sites in Taiwan (n=22) were tested with a detection limit of 1000 cells/mL [89]. Similarly, qPCR was developed to detect CYNs for targeting rpoC1 and cyrJ genes of C. raciborskii species which showed strong linearity between 10 2 and 10 6 copies per reaction.…”