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

Content Provider	PubMed Central
Author	Corinne, Audemard Reece, Kimberly S. Burreson, Eugene M.
Copyright Year	2004
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.
Related Links	http://dx.doi.org/10.1128/aem.70.11.6611-6618.2004
Ending Page	6618
Page Count	8
Starting Page	6611
File Format	PDF
ISSN	00992240
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	11
Volume Number	70
Language	English
Publisher	American Society for Microbiology
Publisher Date	2004-11-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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