A novel duplex qPCR assay for stepwise detection of multiple Perkinsea protistan infections of amphibian tissues

Type Article
Date 2021-03
Language English
Author(s) Smilansky VanessaORCID1, Chambouvet AurelieORCID2, Reeves Mari3, Richards Thomas A.ORCID4, Milner David S.ORCID4
Affiliation(s) 1 : Living Systems Institute and Biosciences, University of Exeter, Stocker Road, Exeter, Devon EX4 4QD, UK
2 : CNRS, Univ Brest, IRD, Ifremer, LEMAR, Plouzané, France
3 : US Fish and Wildlife Service, Pacific Islands Fish and Wildlife Office, Honolulu, Hawai'i
4 : Department of Zoology, University of Oxford, 11a Mansfield Road, Oxford OX1 3SZ, UK
Source Royal Society Open Science (2054-5703) (The Royal Society), 2021-03 , Vol. 8 , N. 3 , P. 202150 (8p.)
DOI 10.1098/rsos.202150
Keyword(s) frog disease, alveolate parasites, quantitative PCR, Perkinsea, NAG01
Abstract

Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested the existence of two clades of amphibian Perkinsea: a putatively non-pathogenic clade linked to asymptomatic infections of tadpoles in Africa, Europe and South America, and a putatively pathogenic clade linked to disease and mass mortality events of tadpoles in North America. Here, we describe the development of a duplex TaqMan qPCR assay to detect and discriminate between rDNA sequences from both clades of Perkinsea in amphibian tissues. The assay uses a single primer pair to target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared between the two clades, and two dual-labelled probes to target a region within this fragment that is diagnostic for each clade. This assay enables rapid screening for each of the two Perkinsea groups, allowing for detection, primarily of the phylogenetic group associated with disease outbreaks, and secondarily for the phylogenetic group with no current disease relationship identified. Incorporation of our novel qPCR assay into the routine surveillance of amphibian populations will allow for the assessment of the incidence of each protist clade, thereby providing an improved understanding of Perkinsea infection pervasiveness and a method to underpin future conservation planning.

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How to cite 

Smilansky Vanessa, Chambouvet Aurelie, Reeves Mari, Richards Thomas A., Milner David S. (2021). A novel duplex qPCR assay for stepwise detection of multiple Perkinsea protistan infections of amphibian tissues. Royal Society Open Science, 8(3), 202150 (8p.). Publisher's official version : https://doi.org/10.1098/rsos.202150 , Open Access version : https://archimer.ifremer.fr/doc/00685/79758/