Real-Time PCR for Detection and Quantification of the Protistan Parasite Perkinsus marinus in Environmental Waters

Abstract

The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genusspecific primers. Detection of DNA concentrations as low as the equivalent of 3.3 ؋ 10 ؊2 cell per 10-l reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.Perkinsus species are parasites of marine molluscs that can have a severe pathogenic effect on their hosts and cause significant economic losses. One of the most detrimental species in this group is Perkinsus marinus (27), a parasite of the eastern oyster, Crassostrea virginica (Gmelin). Since the 1950s, P. marinus has been responsible for severe mortalities among C. virginica populations along the mid-Atlantic region of the United States (1, 7).P. marinus is waterborne and directly transmitted from oyster to oyster (26). Experimentally, transmission of P. marinus is dose dependent and all three known parasite life stages, trophozoite, prezoosporangia, and zoospore, have been shown to induce infection in oysters (1,26,31,41). However, studies of transmission dynamics in the environment have been hindered by the inability to detect free-living stages of the parasite in water. Traditionally, detection of P. marinus in oysters has involved histology or culture of oyster tissues in fluid thioglycolate medium (FTM) (33, 34), and the latter is still the most commonly used diagnostic test to determine infection prevalence and intensity in oysters. The major drawbacks of the FTM assay are its lack of species specificity and its inability to detect low-intensity infections corresponding to fewer than 1,000 P. marinus cells per g of wet oyster tissue (9)....

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“…Quantitative polymerase chain reaction (qPCR) assays now offer advantages of quantifying infection intensities [49][50][51][52], with new tools already appearing on the horizon in the application of proteomic methods such as matrix-assisted laser desorption time-of-flight mass spectrometry [53]. We accept PCR assays as having lived up to promises of improved sensitivity and specificity relative to older methods, which in many cases were based on microscopic methods, including paraffin histopathology for haplosporidians, cytological evaluation of stained haemolymph preparations or tissue imprints for Bonamia and Marteilia species, and Ray's fluid thioglycollate method [54] for Perkinsus parasites (table 1).…”

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