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- Title
Quantitative real-time PCR detection of a harmful unarmoured dinoflagellate, Karlodinium australe ( Dinophyceae).
- Authors
Kon, Nyuk Fong; Lau, Winnie L. S.; Hii, Kieng Soon; Law, Ing Kuo; Teng, Sing Tung; Lim, Hong Chang; Takahashi, Kazuya; Gu, Haifeng; Lim, Po Teen; Leaw, Chui Pin
- Abstract
SUMMARY We investigated a harmful algal bloom ( HAB) associated with the massive fish kills in Johor Strait, Malaysia, which recurred a year after the first incident in 2014. This incident has urged for the need to have a rapid and precise method in HAB monitoring. In this study, we develop a SYBR green-based real-time PCR (q PCR) to detect the culpable dinoflagellate species, Karlodinium australe. Species-specific q PCR primers were designed in the gene region of the second internal transcribed spacer of the ribosomal RNA gene (r DNA). The species specificity of the primers designed was evaluated by screening on the non-target species ( Karlodinium veneficum, Takayama spp., and Karenia spp.) and no cross-detection was observed. The extractable gene copies per cell of K. australe determined in this study were 19 998 ± 505 ( P < 0.0001). Estimation of cell densities by qPCR in the experimental spiked samples showed high correlation with data determined microscopically ( R2 = 0.93). Using the qPCR assay developed in this study, we successfully detected the 2015 bloom species as K. australe. Single-cell PCR and r DNA sequencing from the field samples further confirmed the finding. With the sensitivity as low as five cells, the q PCR assay developed in this study could effectively and rapidly detect cells of K. australe in the environmental samples for monitoring purpose.
- Subjects
JOHOR Strait; DINOFLAGELLATES; ALGAL blooms; FISH kills; POLYMERASE chain reaction
- Publication
Phycological Research, 2017, Vol 65, Issue 4, p291
- ISSN
1322-0829
- Publication type
Article
- DOI
10.1111/pre.12186