Design and validation of universal and taxon-specific 16S rRNA qPCR primers for detection of freshwater harmful algal bloom-forming cyanobacteria

Freshwater harmful algal bloom-forming cyanobacteria have become an increasingly prominent global concern from both environmental and human health perspectives. To enable and facilitate timely decision making in taking preventative and mitigative measures, rapid, accurate, sensitive and quantitative tools are needed for the detection and monitoring of toxin-producing cyanobacteria. Here we report the development of taxon-specific quantitative polymerase chain reaction (qPCR) primers capable of distinguishing 10 cyanobacterial genera or clade from non-target groups as well as a new set of universal primers capable of amplifying all cyanobacteria species. When evaluated by 4 stringent metrics and primer-template mismatches, these de novo designed qPCR primers outperformed published primers in amplifying the 16S rRNA gene of their target strains among the collection of 16 in-house cyanobacterial strains belonging to 10 genera. The 10 best-performing designed primers were validated using field samples from three field locations with historically documented HAB events. The field validation results corroborated with both microscopic observations and Nanopore sequencing of full-length 16S amplicons. Our study demonstrated the effectiveness of our design-screen-evaluation-validation pipeline in developing taxon-specific qPCR primers for detecting and quantifying group-specific target populations and their promising application to field HABs samples. With the advancement of massive parallel sequencing technologies and bioinformatic tools, a community-wide 16S full-length sequencing run can provide a panoramic view of the genetic diversity and site-specific variant info about the target taxa of interest, enabling the fast development of new taxon-specific qPCR assays and their refinement or modification to adapt to site-specific genetic variations in field samples.