Innovative multiplex qPCR method for rapid and reliable detection of microcystin-producing genes during harmful algal...

Cyanobacterial harmful algal blooms (cyanoHABs) have the potential to produce cyanotoxins, which pose significant health risks to both humans and animals. The gold standard methods for monitoring cyanoHABs involve enzyme-linked immunosorbent assay (ELISA), liquid chromatography combined with triple quadrupole mass spectrometry (LC-MS/MS) and manual cell counting under microscopy. However, these techniques, while effective, are costly and time-consuming, which may not be optimal for timely decision-making to safeguard public health. Quantitative polymerase chain reaction (qPCR) offers a complementary approach that serves as an indicator of the potential for toxin production. It provides accurate results with a rapid turnaround time and high throughput capacity, and greater affordability. To assess the reliability of qPCR in predicting toxin production and determining when toxin levels exceed recreational advisory thresholds, we conducted experiments utilizing two DNA extraction methods for qPCR testing: RapidDNA and ClassicDNA. Sampling was conducted across nine water bodies in Utah throughout the recreational season from June 1 to October 31, 2023. We targeted cyanotoxin-encoding genes mainly associated with microcystins, the dominant cyanotoxin reported for these water bodies, for qPCR analysis. Toxin levels were measured using both ELISA and LC-MS/MS with cyanobacteria cell counting conducted as a reference. Out of nine water bodies studied, cyanoHABs were detected in five (i.e., Utah Lake, and Deer Creek, Echo, Schofield, and Pineview Reservoirs). Analysis of the data revealed a significant linear relationship between both the qPCR results of mcyE (associated with microcystin production) obtained from RapidDNA and ClassicDNA methods, and the levels of microcystins measured by ELISA and LC-MS/MS. RapidDNA qPCR methods offer a potential warning tool for indicating toxin production during blooming events, though this method is not suitable for determining risk during the pre-blooming period. Conversely, ClassicDNA methods can be utilized during the pre-blooming period to prepare for potential blooms. These results provide insight into the genetic potential of blooms around the state to produce microcystins. Findings can be implemented in both Recreational Water Quality and Drinking Water programs nationally.