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

Vanessa Smilansky,Mari Reeves,Thomas A Richards,Aurelie Chambouvet,David S Milner

doi:10.1098/rsos.202150

Vanessa Smilansky, Mari Reeves + Show 3 more

Open Access

https://doi.org/10.1098/rsos.202150

Copy DOI

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.

Highlights

Tadpole mass mortality events (MMEs) associated with an alveolate protist in the phylum Perkinsea have been reported across a broad geographic range within the USA [1,2,3,4,5]
We presume that either the amount of target DNA in G2.13 is less than the lower limit of detection (LoD), that the sample has degraded from the time when it was screened for novel alveolate group 01 (NAG01) DNA by Chambouvet et al [6], or that PCR inhibitors are present in the tadpole tissue that limit the detection of NAG01a
The low amount of target DNA contained by putatively asymptomatic samples is important to consider when applying the duplex assay, as our results suggest that large amounts of pathogenic Perkinsea clade (PPC) template can ‘overwhelm’ the signal emitted by the NAG01a-c template

Summary

Introduction

Tadpole mass mortality events (MMEs) associated with an alveolate protist in the phylum Perkinsea have been reported across a broad geographic range within the USA [1,2,3,4,5]. NAG01 includes three clades (NAG01a-c) known to infect tadpoles from a broad range of taxa without causing any identifiable gross or tissue-level symptoms of disease [6], and a discrete clade, designated pathogenic Perkinsea clade (PPC), which is associated with severe Perkinsea infection (SPI) [2]. Given the additional uncertainty regarding the impact of NAG01a-c infections on tadpole populations [6,7], this led us to develop a method which would allow us to investigate the prevalence of the wider NAG01 group. We describe the development of a rapid, sensitive, duplex TaqMan qPCR assay to detect and discriminate PPC and NAG01a-c Perkinsea. We demonstrate that this assay can be performed on handheld qPCR instruments, opening up the possibility for rapid in-the-field testing of NAG01 infections in wild and captive tadpole populations

Methods

Discussion

Conclusion