Affiliation:
1. Virginia Institute of Marine Science, School of Marine Science, College of William and Mary, Gloucester Point, Virginia
Abstract
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
genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 × 10
−2
cell per 10-μl 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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
103 articles.
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