Pooling skin swabs does not inhibit qPCR detection of amphibian chytrid infection.

Joana Sabino-Pinto,An Martel,Sebastian Steinfartz,Frank Pasmans,Miguel Vences

doi:10.1371/journal.pone.0214405

Joana Sabino-Pinto, An Martel + Show 3 more

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https://doi.org/10.1371/journal.pone.0214405

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Abstract

Immediate and reliable pathogen detection in large numbers of samples is essential in wildlife disease monitoring and is often realized by DNA-based techniques. Pooling samples increases processing efficiency and reduces processing costs, and has been suggested as a viable technique for quantitative PCR detection of fungal amphibian pathogens of the genus Batrachochytrium. For these fungi, this diagnostic method has been validated by in vitro set ups that provided controlled test conditions but did not take into account potential effects from amphibian skin compounds (e.g. skin secretions and Microbiota) on the approach. Some of these skin compounds are known to cause PCR inhibition in single sample applications and could lead to false negative reactions and thereby hamper pathogen detection. In this study we examined the effect of skin compounds on the pooled extraction method by swabbing individuals of seven amphibian species (one Anura and six Caudata) prior to the inoculation of the swabs with chytrid zoospores. For each species, swabs were extracted in pools of different sizes (from one to four swabs) with only one swab per pool being inoculated with zoospores. There were no significant differences regarding the ability to detect zoospores when comparing pool sizes for any species, with a tendency for more false negatives when the inoculated swab had been inoculated with a single zoospore. This study provides further in vivo evidence for the viability of the pooled extraction method for DNA-based detection of pathogens.

Highlights

Emerging infectious diseases (EIDs) are diseases that are caused by pathogens that have recently expanded their range, either geographically and/or in host species [1]
All ethical procedures were followed; skin swabs were collected at Ghent University where no permit is required to collect such samples
The size of the pool had no direct effect on the detectability of the B. salamandrivorans (Bsal)– fungus (GLM: χ2(1,3) = 0.14, p-value = 0.704; Table 1), or on the amount of Bsal zoospores detected (LM: F(1,71) = 2.10, p-value = 0.152; Table 1)

Summary

Introduction

Emerging infectious diseases (EIDs) are diseases that are caused by pathogens that have recently expanded their range, either geographically and/or in host species [1]. They can be divided in three groups: 1) EIDs invading new hosts; 2) EIDs caused by a mutated pathogen (increased pathogenicity in the same host); and 3) EIDs invading new geographical areas (invasive species) [2]. When tackling EIDs, the first step is the determination of the pathogen distribution, often with molecular (DNA-based) techniques. This requires the collection and processing of large amounts of samples, with time of processing being especially important when sampling for highly infectious and lethal pathogens

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