Application of real-time PCR assay for detection and quantification of bloom-forming diatom Chaetoceros tenuissimus Meunier

Viral infection of the bloom-forming diatom Chaetoceros tenuissimus was discovered in 2008, and is now assumed to have a significant influence on the dynamics of C. tenuissimus populations in natural environments. Enumeration of C. tenuissimus and its viruses is essential when examining the host-virus relationship in situ; however, the diatom species is so small in size that its identification and counting by optical microscope is almost impossible. To resolve this problem, we have developed a TaqMan-based real-time polymerase chain reaction (PCR) method for detection and quantification of C. tenuissimus. We designed primers and a TaqMan probe to target the D1 region of its 28S ribosomal RNA (rRNA) gene; the established real-time PCR was specific at the species level by testing 41 microalgal strains including C. tenuissimus. Tris-EDTA buffer-based boiling method was shown to be efficient for extracting DNA from filter-trapped C. tenuissimus cells in this study. The detection range of the established TaqMan-based real-time PCR method for C. tenuissimus was 10¹ to 10⁶ cells collected on a filter; the method was applicable for C. tenuissimus cells accompanied with natural microorganisms.