TaqMan registered MGB real-time PCR approach to quantification ofPerkinsus marinus and Perkinsus spp. in oysters

摘要

Several molecular diagnostic assays have been developed in an attempt to replace the traditional Ray's Fluid Thiogly-collate 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 registered 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 (R super(2) greater than or equal to 0.99) between parasite cell density and real-time PCR threshold cycle (C sub(T)) with amplification efficiencies greater than or equal to 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 (R super(2) > 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.