Optimizing, validating, and field testing a multiplex qPCR for the detection of amphibian pathogens

Optimizing, validating, and field testing a multiplex qPCR for the detection of amphibian pathogens

Amphibian populations worldwide are facing numerous threats, including the emergence and spread of infectious diseases. In the past 2 decades, Batrachochytrium dendrobatidis (Bd), a parasitic fungus, and a group of viruses comprising the genus Ranavirus have become widespread and resulted in mass mo...

Full description

Saved in:
Bibliographic Details
Published in:Diseases of aquatic organisms Vol. 129; no. 1; pp. 1 - 13
Main Authors: Standish, Isaac, Leis, Eric, Schmitz, Noel, Credico, Jeena, Erickson, Sara, Bailey, Jennifer, Kerby, Jacob, Phillips, Kenneth, Lewis, Teresa
Format: Journal Article
Language:English
Published: Germany Inter-Research Science Center 19-06-2018
Subjects:
Batrachochytrium
Diagnostic systems
Fungi
Genes
Genomes
Infections
Infectious diseases
Multiplexing
Optimization
Parasitic diseases
Pathogens
Polymerase chain reaction
Populations
Reproducibility
Reptiles & amphibians
Viruses
Wildlife
Wildlife habitats
Ranavirus
Amphibian pathogens
Batrachochytrium dendrobatidis
Multiplex qPCR
Batrachochytrium salamandrivorans
Wisconsin
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Amphibian populations worldwide are facing numerous threats, including the emergence and spread of infectious diseases. In the past 2 decades, Batrachochytrium dendrobatidis (Bd), a parasitic fungus, and a group of viruses comprising the genus Ranavirus have become widespread and resulted in mass mortality events and extirpations worldwide. In 2013, another novel fungus, B. salamandrivorans (Bsal), was attributed to dramatic declines in populations of fire salamander Salamandra salamandra in the Netherlands. Experimental infections demonstrated that Bsal is highly pathogenic to numerous salamander genera. In an effort to prevent the introduction of Bsal to North America, the US Fish and Wildlife Service (USFWS) listed 201 salamander species as injurious wildlife under the Lacey Act. To determine infection status and accurately assess amphibian health, the development of a sensitive and specific diagnostic assay was needed. We describe the optimization and validation of a multiplex quantitative polymerase chain reaction (qPCR) protocol for the simultaneous detection of Bd, Bsal, and frog virus 3-like ranaviruses. A synthetic genome template (gBlock®) containing the target genes from all 3 pathogens served as the positive control and allowed accurate quantification of pathogen genes. The assay was validated in the field using an established non-lethal swabbing technique to survey local amphibian populations throughout a range of habitats. This multiplex qPCR demonstrates high reproducibility, sensitivity, and was capable of detecting both Bd and ranavirus in numerous locations, species, and life stages. Bsal was not detected at any point during these sampling efforts.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:0177-5103
1616-1580
DOI:10.3354/dao03230