Development of single-cell PCR methods for the Raphidophyceae

Many Raphidophytes are important algal bloom-forming species. Morphology-based identification of these species is often ambiguous, however, as many species are very similar in shape and size. To accurately detect the presence of these species in pre-bloom conditions, single-cell PCR is probably the most rapid and convenient method. However, direct single-cell PCRs with conserved primers are apparently not effective, probably due to the impermeability of the cell wall. We report here an effective detergent-based pre-PCR cell lysis method, which turned out to be a critical step for effective single-cell PCR of the Raphidophytes. Two PCR-based methods, nested SC-PCR and SC-RAPD, were evaluated. The nested SC-PCR involves two consecutive PCRs, the first of which is performed with the D1 and D2 primers (external primers) resulting in an amplification of a partial LSU rRNA gene. The second amplification is performed with primers targeting the LSU domain and specifically annealing to Chattonella ovata and Chattonella marina only. The SC-RAPD performed with the established random primers, RP1-RP4, produced unique haplotypes that could be exploited to differentiate the two Chattonella species. The assay was demonstrated to be sensitive, with the lowest detection limit of a single Raphidophyceae cell. The method developed is a valuable tool for the study of intra-specific variations of the Raphidophytes and represents a platform for further development of species-specific SC-RAPD for all members of the Raphidophyceae.