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1 March 2009 Development and Optimization of Quantitative PCR Assays to Aid Ostrea lurida Carpenter 1864 Restoration Efforts
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The Olympia oyster (Ostrea lurida) is a prime candidate for the development of a rapid, high throughput, species-specific larval identification and quantification assay. We developed O. lurida specific DNA primers and a fluorescently labeled probe that amplify a mitochondrial DNA region cytochrome oxidase 1 subunit (COI) to use in quantitative polymerase chain reaction (qPCR). We also developed qPCRs for the specific detection of Neotrypaea californiensis and Upogebia pugettensis, two burrowing shrimp species that have been shown to have a negative effect on oyster bed habitat. The primer and probes amplified only the target organisms. Using standard curves constructed from known quantities of larvae, we are able to rapidly and accurately identify and estimate unknown quantities of larvae. In blind tests, direct counts of O. lurida larvae did not significantly differ from qPCR estimates. DNA was fully liberated from up to 80 O. lurida larvae, and 10 N. californiensis larvae; PCRs were not inhibited as demonstrated by an internal positive control multiplexed into the qPCRs. Genetic based assays are an extremely useful method for sorting complex plankton samples that can be more time and cost effective than traditional microscopy techniques. Our qPCR assay may prove to be a valuable tool to monitor O. lurida restoration site productivity as well as increase our understanding of this critical life history stage.

Nathan A. Wight, Jason Suzuki, Brent Vadopalas, and Carolyn S. Friedman "Development and Optimization of Quantitative PCR Assays to Aid Ostrea lurida Carpenter 1864 Restoration Efforts," Journal of Shellfish Research 28(1), 33-41, (1 March 2009).
Published: 1 March 2009

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