Development and validation of a specific real-time PCR assay for the detection of the parasite Perkinsus olseni

• Published in: Journal of invertebrate pathology Vol. 169; p. 107301
• Main Authors: Ríos, R., Aranguren, R., Gastaldelli, M., Arcangeli, G., Novoa, B., Figueras, A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2020
• Subjects: Alveolata - isolation & purification; Animals; Bivalvia - parasitology; Host-Parasite Interactions; Perkinsus; qPCR; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - veterinary; Reproducibility of Results; Ruditapes decussatus; Ruditapes phillipinarum; Thioglycollate; Ct; Thioglycollate; NPV; Ruditapes phillipinarum; PPV; qPCR; RFTM; PCR-RFLP; O.I.E; Perkinsus; Ruditapes decussatus
• ISSN: 0022-2011; 1096-0805
• DOI: 10.1016/j.jip.2019.107301

[Display omitted] •A specific diagnosis of Perkinsus olseni was obtained with a new qPCR assay.•The results of the real-time PCR assay were sensitive and reproducible.•No false negatives were obtained with the qPCR assay compared with traditional methods.•Gill tissue provided more accurate qPCR results than those of hemolymph samples. Perkinsus olseni is a protozoan parasite that infects a wide variety of molluscs worldwide, causing economic losses in the aquaculture sector. In the present study, a quantitative PCR (qPCR) assay was developed for the detection and quantification of P. olseni in clam gill tissue and hemolymph (Ruditapes philippinarum and R. decussatus), and the results were compared with those of the standard diagnostic methods recommended by the O.I.E. (World Organisation for Animal Health): Ray's fluid thioglycollate culture method (RFTM), a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay and histopathology. The efficiency, sensitivity and reproducibility of the newly described qPCR assay were also determined. The highest prevalence was detected using the qPCR assay, and the strongest linear correlation was obtained between the RFTM infection levels and the threshold cycle (Ct) number from the gill tissue. Although better results were obtained from gill than from the hemolymph in the qPCR assays, especially with lower infection levels of the parasite, a significant linear correlation was observed between Ct values from the gill and hemolymph. The qPCR assay that was developed in this study showed high sensitivity, specificity and reproducibility for the detection and quantification of P. olseni.