Translator Disclaimer
1 August 2006 TAQMAN® MGB REAL-TIME PCR APPROACH TO QUANTIFICATION OF PERKINSUS MARINUS AND PERKINSUS SPP. IN OYSTERS
J. D. GAUTHIER, C. R. MILLER, A. E. WILBUR
Author Affiliations +
Abstract

Several molecular diagnostic assays have been developed in an attempt to replace the traditional Ray's Fluid Thioglycollate Medium (RFTM) assay for detection and quantification of Perkinsus marinus in oysters. Real-time PCR technology is a state-of-the-art method currently used to diagnose disease intensity in vertebrates. We developed a simple (two-reagent) real-time PCR assay to quantify P. marinus (PMAR) and Perkinsus spp. (PERK) in oysters, using TaqMan® assays designed with Minor Groove Binder (MGB) probes on an Applied Biosystems 7500 Real-Time PCR System. Both PERK and PMAR assays demonstrate strong correlations (R2 ≥ 0.99) between parasite cell density and real-time PCR threshold cycle (CT) with amplification efficiencies ≥99%. The PERK assay results in similar amplification plots for the three species tested (P. marinus, P. olseni and P. chesapeaki), whereas the PMAR assay detects only P. marinus. A strong correlation (R2 > 0.90) was found between infection level determined by the traditional RFTM method and quantification by real-time PCR, based on internal standards prepared from P. marinus spiked oyster tissue. The PCR assays also detected Perkinsus in oysters diagnosed as negatives using the traditional method, suggesting that the described assay may be more sensitive. These assays provide a nonsubjective, specific and accurate quantification of P. marinus in oyster tissues and thus could potentially replace the traditional method in some applications.

J. D. GAUTHIER, C. R. MILLER, and A. E. WILBUR "TAQMAN® MGB REAL-TIME PCR APPROACH TO QUANTIFICATION OF PERKINSUS MARINUS AND PERKINSUS SPP. IN OYSTERS," Journal of Shellfish Research 25(2), 619-624, (1 August 2006). https://doi.org/10.2983/0730-8000(2006)25[619:TMRPAT]2.0.CO;2
Published: 1 August 2006
JOURNAL ARTICLE
6 PAGES


SHARE
ARTICLE IMPACT
RIGHTS & PERMISSIONS
Get copyright permission
Back to Top