Development and Validation of a Real-Time Quantitative PCR Assay for the Detection and Quantification ofPerkinsus marinusin the Eastern Oyster,Crassostrea virginica

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.

Development of real-time PCR assays for discrimination and quantification of two Perkinsus spp. in the Manila clam Ruditapes philippinarum

“…(Table 1) using a 10% (w/w) Chelex (Bio-Rad Laboratories Japan) resin solution (Barber & Erdmann 2000, De Faveri et al 2009). Briefly, Chelex solution was prepared by suspending molecular biology grade Chelex 100 resin (200−400 dry mesh, 75−150 µm wet beads; Biorad-Laboratories Japan) in ultrapure water.…”

Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica

“…The r 2 values of 0.636 and 0.797 for C q values versus categorical and density measures, respectively, are modest compared to that found for cloned Haplosporidium nelsoni target region or the 0.826 to 0.99 for calibration curves from cultured Perkinsus marinus (Yarnall et al 2000, Audemard et al. 2004, Gauthier et al 2006, De Faveri et al 2009) and quahog parasite unknown (QPX; Lyons et al 2006, Liu et al 2009). Nevertheless, they are significant considering the challenges of accurate parasite quantification in histological samples and the uneven distribution of H. nelsoni plasmodia in the tissues, especially in early infections when parasites are localized in the gill or sparsely scattered in the visceral mass (Ford & Haskin 1982 Table 4.…”

“…These limitations have prompted interest in real-time or quantitative PCR (qPCR) assays, which have followed a trajectory similar to end-point PCR, from initial development as a diagnostic tool (Day et al 2000, Yarnall et al 2000, Audemard et al 2004, Gauthier et al 2006, Lyons et al 2006, De Faveri et al 2009) to application for understanding the ecology of the parasite in the environment, and mechanisms of host−pathogen interactions (Audemard et al 2006, Marty et al 2006, Gast et al 2008, Liu et al 2009). These applications have been made possible by the fact that qPCR assays permit the absolute or relative quantification of the target.…”

“…These applications have been made possible by the fact that qPCR assays permit the absolute or relative quantification of the target. For species of parasites that can be propagated in vitro, a qPCR assay can be calibrated using counts of cultured parasites (Yarnall et al 2000, Audemard et al 2004, Gauthier et al 2006, De Faveri et al 2009) to convert relative to absolute abundance. The inability to propagate some important bivalve pathogens complicates the development of qPCR assays for those pathogens.…”

The ecology, evolution, impacts and management of host–parasite interactions of marine molluscs

“…The molecular revolution has produced new techniques that allow rapid diagnosis of pathogens (e.g., Reece et al, 1997Reece et al, , 2008De Faveri et al, 2009;Wight et al, 2009;Wilbur et al, 2012). Whereas in the past, epidemiologists to a large extent relied on often ineffective culturing techniques and histology to identify marine pathogens, species-specific DNA probes now enable screening for a broad range of pathogens (e.g., Harvell et al, 1999).…”