Development of duplex TaqMan-based real-time PCR assay for the simultaneous detection of Perkinsus olseni and P. chesapeaki in host Manila clam tissue samples

Type Article
Date 2021-09
Language English
Author(s) Itoïz Sarah1, Perennou Morgan1, Mouronvalle Clara1, 2, Derelle Evelyne1, Le Goïc Nelly1, Bidault Adeline1, 5, de Montaudouin Xavier3, Arzul IsabelleORCID4, Soudant Philippe1, Chambouvet Aurélie1
Affiliation(s) 1 : Univ Brest, CNRS, IRD, Ifremer, LEMAR, F-29280 Plouzané, France
2 : EPHE, PSL Research University, UPVD, CNRS, USR 3278 CRIOBE, Perpignan F-66360, France
3 : Univ. Bordeaux, CNRS, EPOC, EPHE, UMR 5805, Station Marine, F-33120 Arcachon, France
4 : IFREMER, Laboratory of Genetics and Pathology, Av de Mus de Loup-17390, La Tremblade, France
5 : Univ Brest, CNRS, IRD, Ifremer, LEMAR, F-29280 Plouzané, France
Source Journal Of Invertebrate Pathology (0022-2011) (Elsevier BV), 2021-09 , Vol. 184 , P. 107603 (12p.)
DOI 10.1016/j.jip.2021.107603
WOS© Times Cited 1
Keyword(s) Alveolata, Co-infection, Molluscs, Inhibitory effect, Ruditapes philippinarum, qPCR
Abstract

The aetiological agent Perkinsus olseni is globally recognised as a major threat for shellfish production considering its wide geographical distribution across Asia, Europe, Australia and South America. Another species, Perkinsus chesapeaki, which has never been known to be associated with significant mortality events, was recently detected along French coasts infecting clam populations sporadically in association with P. olseni. Identifying potential cryptic infections affecting Ruditapes philippinarum is essential to develop appropriate host resource management strategies. Here, we developed a molecular method based on duplex real-time quantitative PCR for the simultaneous detection of these two parasites, P. olseni and P. chesapeaki, in the different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the rest of the soft body. We firstly checked the presence of possible PCR inhibitors in host tissue samples. The qPCR reactions were inhibited depending on the nature of the host organ. The mantle and the rest of the soft body have a high inhibitory effect from threshold of host gDNA concentration of 2 ng.µL−1, the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL−1, and the gills and digestive gland do not show any inhibition of the qPCR reaction even at the highest host gDNA concentration of 20 ng.µL−1. Then, using the gills as a template, the suitability of the molecular technique was checked in comparison with the Ray’s Fluid Thioglycolate Medium methodology recommended by the World Organisation for Animal Health. The duplex qPCR method brought new insights and unveiled cryptic infections as the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in contrast to the RFTM diagnosis. The development of this duplex qPCR method is a fundamental work to monitor in situ co-infections that will lead to optimised resource management and conservation strategies to deal with emerging diseases.

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Itoïz Sarah, Perennou Morgan, Mouronvalle Clara, Derelle Evelyne, Le Goïc Nelly, Bidault Adeline, de Montaudouin Xavier, Arzul Isabelle, Soudant Philippe, Chambouvet Aurélie (2021). Development of duplex TaqMan-based real-time PCR assay for the simultaneous detection of Perkinsus olseni and P. chesapeaki in host Manila clam tissue samples. Journal Of Invertebrate Pathology, 184, 107603 (12p.). Publisher's official version : https://doi.org/10.1016/j.jip.2021.107603 , Open Access version : https://archimer.ifremer.fr/doc/00711/82263/