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Development and validation of a real-time quantitative PCR assay for the detection and quantification of Perkinsus marinus in the Eastern oyster, Crassostrea virginica

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dc.contributor.author De Faveri, Jacquelin
dc.contributor.author Smolowitz, Roxanna M.
dc.contributor.author Roberts, Steven B.
dc.date.accessioned 2010-04-14T14:19:18Z
dc.date.available 2010-04-14T14:19:18Z
dc.date.issued 2009-08
dc.identifier.citation Journal of Shellfish Research 28 (2009): 459-464 en_US
dc.identifier.uri http://hdl.handle.net/1912/3241
dc.description Author Posting. © National Shellfisheries Association, 2009. This article is posted here by permission of National Shellfisheries Association for personal use, not for redistribution. The definitive version was published in Journal of Shellfish Research 28 (2009): 459-464, doi:10.2983/035.028.0306. en_US
dc.description.abstract Perkinus marinus causes a devastating disease, known as Dermo, in the Eastern oyster Crassostrea virginica. Routine detection of the disease is traditionally accomplished by the use of the Ray/Makin assay, using Fluid Thioglycollate Medium (RFTM). A simple real-time quantitative PCR assay was developed as a diagnostic tool to detect and quantify P. marinus, to complement and serve as an alternate to the RFTM method. Using a dual-labeled probe approach, a sensitive assay was designed to accurately detect a range of one to several thousand P. marinus organisms present in oyster tissues. A simple extraction method was used to increase throughput of the assay. Cultured P. marinus cells were quantified prior to DNA extraction, generating a standard curve and allowing cell counts to be derived from PCR cycle threshold values. Direct comparison of the RFTM and real-time PCR methods was accomplished by using tissue samples from the same oyster for both tests. Plotting cycle threshold values against the known Mackin index value generated a standard curve with a coefficient of regression of 0.9. Our results indicate that correlations could be made between this molecular based approach and traditional methods, allowing results generated from the PCR assay to be easily translated into the understood Mackin scale. en_US
dc.format.mimetype application/pdf
dc.language.iso en_US en_US
dc.publisher National Shellfisheries Association en_US
dc.relation.uri http://dx.doi.org/10.2983/035.028.0306
dc.subject Dermo en_US
dc.subject Perkinsus marinus en_US
dc.subject Real-time PCR en_US
dc.subject RFTM en_US
dc.subject Eastern oyster en_US
dc.subject Crassostrea virginica en_US
dc.title Development and validation of a real-time quantitative PCR assay for the detection and quantification of Perkinsus marinus in the Eastern oyster, Crassostrea virginica en_US
dc.type Article en_US
dc.identifier.doi 10.2983/035.028.0306


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