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

Kensuke Toyoda, Keizo Nagasaki, Yuji Tomaru

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)56-61
Number of pages6
JournalPlankton and Benthos Research
Volume5
Issue number2
DOIs
Publication statusPublished - 2010 Jul 15

Keywords

  • Chaetoceros tenuissimus
  • Detection
  • Diatom
  • Enumeration
  • TaqMan-based real-time PCR

ASJC Scopus subject areas

  • Oceanography
  • Ecology, Evolution, Behavior and Systematics
  • Aquatic Science
  • Ecology

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