Improved quantitative real-time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard

Quantitative real-time PCR (QPCR) is a powerful and sensitive method for quantitative detection of microorganisms. Application of this methodology for enumeration of harmful algal bloom (HAB) species has the potential to revolutionize our approach to HAB research, making it possible to identify correlations between cell abundances and factors that regulate bloom dynamics. Its application to ecological studies, however, has produced mixed results. QPCR assays typically rely on the generation of standard curves from plasmids or laboratory cultures that may be unrealistic when compared to amplification of DNA extracted from field samples. In addition, existing methods often fail to incorporate controls to assess variability in extraction and amplification efficiencies, or include controls that are sequence-specific and preclude the investigation of multiple species. Here, we describe the development and rigorous analysis of QPCR assays for two HAB species, Chattonella subsalsa and Heterosigma akashiwo, in which we introduce a known concentration of exogenous DNA plasmid into the extraction buffer as a reference standard. Since the target DNA is extracted in the presence of the reference standard, inherent variability in extraction and amplification efficiencies affect both target and standard equally. Furthermore, the reference standard is applicable to QPCR analysis of any microbial species. Using environmental bloom samples as calibrators, we evaluated the accuracy of the comparative Ct method for enumeration of target species in several field samples. Our investigation demonstrates that the comparative Ct method with an exogenous DNA reference standard provides both accurate and reproducible quantification of HAB species in environmental samples.