Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis

Type Article
Date 2009-12
Language English
Author(s) Robert Maeva1, Garcia Celine1, Chollet Bruno1, Lopez-Flores Inmaculada2, Ferrand Sylvie1, Francois Cyrille1, Joly Jean-Pierre1, Arzul IsabelleORCID1
Affiliation(s) 1 : IFREMER, Lab Genet & Pathol, F-17390 La Tremblade, France.
2 : Andalusian Sch Publ Hlth, CIBEResp, Granada 18080, Spain.
Source Molecular and Cellular Probes (0890-8508) (Elsevier), 2009-12 , Vol. 23 , N. 6 , P. 264-271
DOI 10.1016/j.mcp.2009.06.002
WOS© Times Cited 19
Keyword(s) Heart imprints, Ostrea edulis, Quantification, Detection, Real time PCR, Bonamia ostreae
Abstract Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR® Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host–parasite relationship.
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Robert Maeva, Garcia Celine, Chollet Bruno, Lopez-Flores Inmaculada, Ferrand Sylvie, Francois Cyrille, Joly Jean-Pierre, Arzul Isabelle (2009). Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis. Molecular and Cellular Probes, 23(6), 264-271. Publisher's official version : , Open Access version :