Simultaneous enumeration of multiple raphidophyte species by quantitative real-time PCR: capabilities and limitations

Quantitative real-time PCR (QPCR) is a powerful method for species-specific detection and enumeration of harmful algal bloom (HAB) algae, but can be quite time-consuming and expensive for multiple species detection when only a single species is analyzed in each reaction. Rapid enumeration methods would be valuable for the investigation of multiple HAB events such as the mixed blooms of harmful raphidophyte species (Chattonella cf verruculosa, C. subsalsa, and Heterosigma akashiwo) that co-occur in the Delaware Inland Bays (DIB), USA. This technology uses multiple species-specific probes that fluoresce at different wavelengths and allows for the simultaneous detection and enumeration of more than one species, limited only by the detection capabilities of the instrument. The approach can cut analysis time and cost at least in half, depending on the number of species that can be accurately detected and enumerated in the mixture. Here, we compared multiprobing - or using a single primer set with species-specific probes - to multiplexing, in which species-specific primers and probes are used, targeting the 18S rDNA of 3 raphidophyte species and an internal standard. Although both methods were effective for multispecies detection compared to simplex results, multiplexing was more accurate than multiprobing for this particular group of species. We investigated the accuracy and sensitivity of multiplex/multiprobe QPCR using plasmids, genomic DNA from cultures, or DNA extracted from environmental samples as template. Smaller amplicon size and sequence heterogeneity between amplicons increased the accuracy of the results. We found that environmental samples of raphidophytes can be successfully multiplexed or multiprobed with only minimal losses in sensitivity. In addition, we explored probe reporter dyes and quenchers for their compatibility, robustness, and reproducibility in multitemplate detection, and we compared and evaluated the capabilities and limitations of this method for detecting and enumerating multiple phytoplankton species simultaneously.