Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR

Abstract

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.

Keywords:

• barnacle
• larvae
• quantitative detection
• quantitative real-time polymerase chain reaction
• species identification
• 12S ribosomal RNA

Acknowledgements

The authors thank Professor T Yamaguchi of Chiba University, Professor R Kado of Kitasato University, Associate Professor K Okamoto of the University of Tokyo and T Ohkawara of the Maisaka Oyster Cultivation Union of Lake Hamana for their assistance in the collection of several barnacle species. The authors also thank Mr O Abe and Mr A Dazai of the Shizugawa Nature Center for their assistance in the sampling of plankton samples, and Dr T Furuta of the Central Research Institute of Electric Power Industry for helpful advice with regard to this paper.

Additional information

Notes on contributors

Kiyotaka Matsumura ^†

† Present address: Department of Biology, Hong Kong University of Science and Technology, Hong Kong