Rapid identification of marine algae (Raphidophyceae) using three-primer PCR amplification of nuclear internal transcribed spacer (ITS) regions from fresh and archived material

Abstract

The internal transcribed spacer region of the nuclear rRNA gene was used to discriminate among the marine raphidophyte species Chattonella antiqua, Chattonella subsalsa, Fibrocapsa japonica, Heterosigma akashiwo, and Olisthodiscus luteus. These microalgae can be difficult to identify from natural water samples, owing to their fragile nature and pleomorphic morphology. Several of these species have been associated with massive fin-fish kills throughout the world, and positive identification is therefore of great economic importance. Species-specific diagnostic polymerase chain reaction (PCR) fragment sizes resulting from three-primer amplification reactions can reproducibly detect as few as five cells per fixed sample and as few as one cell from fresh or frozen samples. Recently, an active area of ecological research has been retrospective studies from archival material to determine population dynamics and species diversity. The PCR primers were successfully used with archived material containing either formalin or Lugol's iodine, as well as with fresh cultures. Because of the existence of other techniques more conducive to monitoring for aquaculture, this assay was developed primarily as a research tool for identification of Raphidophyceae from both cultured and archived field samples where cell integrity has been compromised. It has proved to be a valuable technique for detection and discrimination among marine Raphidophyceae, especially when cells cannot be identified morphologically and fixation methods have destroyed cellular RNA content.