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Single-step nested multiplex PCR to differentiate between various bivalve larvae

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Abstract

A nested multiplex polymerase chain reaction (PCR) assay has been developed that allows the discrimination between six bivalve larvae common to Danish coastal waters (Cerastoderma edule, Macoma balthica, Mytilus edulis, Spisula subtruncata, Ensis americanus and members of the order Myoida). This assay involves the simultaneous use of a pair of general universally targeted 18S rRNA gene primers, five specific 18S rRNA gene targeted oligonucleotide primers internal (nested) to the universal primer pair and one species-specific primer that is not nested (Mya). The specificity of each primer was evaluated in silico, empirically, and verified further by sequencing of amplification products from single larvae collected from plankton samples. Identification of individually isolated bivalve larvae from plankton samples was based on the size of the PCR product produced by the specific primers after visualisation by agarose gel electrophoresis. Preliminary studies indicated that this method was suitable for use with freshly collected and preserved larvae, and is therefore suitable for field application.

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Acknowledgements

We would like to thank T. Møller Pedersen, Dr. A.B. Josefson and Dr. J. Hansen for collecting plankton samples and adult specimens of bivalves. We also would like to thank Dr. G. Høpner Petersen for help in species identification of adult bivalve specimens. A warm thanks goes to everybody in the Frischer laboratory at the Skidaway Institute of Oceanography, to L.J. Rasmussen’s laboratory for technical assistance and to Dr. H.C. Bisgaard for advice during the early stage of the study. This research was supported by the Danish National Science Research Council no. 21010549, the Carlsberg Foundation project no. 990812/10-1177 to B.W.H., and grants from the US National Science Foundation OCE 99-82133 and the US Department of Energy FG02-98ER62531 to M.E.F. The experiments presented in this paper comply with the current law of Denmark.

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  1. Department of Life Sciences and Chemistry, Roskilde University, 4000, Roskilde, Denmark

    J. B. Larsen, L. J. Rasmussen & B. W. Hansen

  2. Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, GA, 31411, USA

    M. E. Frischer

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  1. J. B. Larsen
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  2. M. E. Frischer
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  4. B. W. Hansen
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Correspondence to B. W. Hansen.

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Communicated by M. Kühl, Helsingør

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Larsen, J.B., Frischer, M.E., Rasmussen, L.J. et al. Single-step nested multiplex PCR to differentiate between various bivalve larvae. Marine Biology 146, 1119–1129 (2005). https://doi.org/10.1007/s00227-004-1524-2

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