Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis.
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.
Robert
Maeva
M
Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Laboratoire de Génétique et Pathologie, av de Mus de Loup, 17390 La Tremblade, France.
Garcia
Céline
C
Chollet
Bruno
B
Lopez-Flores
Inmaculada
I
Ferrand
Sylvie
S
François
Cyrille
C
Joly
Jean-Pierre
JP
Arzul
Isabelle
I
D000818:Animals; D018031:Connexin 43 / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D041602:Haplosporida / Q000235:genetics / Q000502:physiology*; D006790:Host-Parasite Interactions; D016014:Linear Models; D049895:Ostrea / Q000254:growth & development / Q000469:parasitology*; D010446:Peptide Fragments / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015800:Protozoan Proteins / Q000235:genetics; D015203:Reproducibility of Results
C541130:ACT1 protein; D018031:Connexin 43; D016054:DNA, Protozoan; D010446:Peptide Fragments; D015800:Protozoan Proteins