Development and validation of a real-time quantitative PCR assay for the detection and quantification of Perkinsus marinus in the Eastern oyster, Crassostrea virginica
Smolowitz, Roxanna M.
Roberts, Steven B.
MetadataShow full item record
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.
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.
Suggested CitationJournal of Shellfish Research 28 (2009): 459-464
Showing items related by title, author, creator and subject.
Light stimulates swimming behavior of larval eastern oysters Crassostrea virginica in turbulent flow Wheeler, Jeanette D.; Luo, Elaine; Helfrich, Karl R.; Anderson, Erik J.; Starczak, Victoria R.; Mullineaux, Lauren S. (2017-05)Planktonic larvae of the eastern oyster Crassostrea virginica are able to regulate their vertical position in the water, but the environmental cues responsible for this regulation, particularly in turbulent settings, remain ...
Desperate planktotrophs : decreased settlement selectivity with age in competent eastern oyster Crassostrea virginica larvae Meyer, Kirstin S.; Wheeler, Jeanette D.; Houlihan, Erin; Mullineaux, Lauren S. (2018-07)For larvae of benthic marine invertebrate species, settlement from planktonic to benthic life is a critical transition. The “desperate larva” concept describes the tendency of larvae to accept suboptimal settlement ...
Estimating connectivity of hard clam (Mercenaria mercenaria) and eastern oyster (Crassostrea virginica) larvae in Barnegat Bay Goodwin, Jacob D.; Munroe, Daphne M.; Defne, Zafer; Ganju, Neil K.; Vasslides, James (MDPI, 2019-06-01)Many marine organisms have a well-known adult sessile stage. Unfortunately, our lack of knowledge regarding their larval transient stage hinders our understanding of their basic ecology and connectivity. Larvae can have ...