Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species

•A multiplex-qPCR method using hydrolysis probes for enumeration of 4 HAB species was developed.•Simplex (S-qPCR) assays were first optimised before being combined for the M-qPCR assay.•Results from M-qPCR assays were comparable to S-qPCR reactions.•The M-qPCR assay provided similar results even when the DNA concentrations varied 104–105-fold.•The assay was tested with spiked natural samples from Danish coastal waters.

Harmful algal blooms (HAB) pose serious economic and health risks worldwide. Current methods of identification require high levels of taxonomic skill and can be highly time-consuming thus limiting sample throughput. So, new rapid and reliable methods for detection and enumeration of HAB species are required. Here we describe a high-throughput, multiplex-qPCR (M-qPCR) method using hydrolysis probe technology for the simultaneous detection of four HAB species commonly found in many coastal areas worldwide: Alexandrium tamarense, Karenia mikimotoi, Karlodinium veneficum and Prymnesium parvum. Primers and probes were species-specific and highly efficient when tested in simplex. Species were then added in succession and the assay conditions adjusted until all four species could be quantitatively evaluated simultaneously. Enumeration accuracy of the M-qPCR assay as a monitoring tool was evaluated using spiked natural environmental samples from Danish coastal waters. Comparison of estimates of cell abundances obtained by the M-qPCR technique with those obtained by light microscopy (Sedgwick Rafter technique) showed no statistically significant difference across a range of concentrations. We were also able to identify and enumerate target cells that would be below the detection limit of light microscopy making this a suitable method for early bloom detection or for low biomass species. With the development of molecular probes for a greater number of algal species M-qPCR will be of great benefit to phytoplankton monitoring programmes and the aquaculture industry worldwide.