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

Area of Science:

• Amphibian pathology
• Wildlife disease diagnostics
• Conservation genetics

Background:

• Amphibian populations face threats from diseases like Batrachochytrium dendrobatidis (Bd) and Ranavirus.
• B. salamandrivorans (Bsal) poses a significant threat to salamander populations, leading to preventative measures like import restrictions.
• Accurate diagnostic tools are crucial for monitoring amphibian health and preventing disease spread.

Purpose of the Study:

• To develop and validate a multiplex quantitative polymerase chain reaction (qPCR) assay.
• To enable simultaneous detection of Bd, Bsal, and ranaviruses.
• To support amphibian health assessments and disease surveillance efforts.

Main Methods:

• Optimization and validation of a multiplex qPCR protocol.
• Utilized a synthetic genome template (gBlock®) for accurate quantification.
• Employed a non-lethal swabbing technique for field-based population surveys.

Main Results:

• The multiplex qPCR assay demonstrated high reproducibility and sensitivity.
• Bd and ranavirus were detected in various amphibian species, locations, and life stages.
• Bsal was not detected in any of the surveyed populations.

Conclusions:

• The developed multiplex qPCR assay is a reliable tool for simultaneous detection of key amphibian pathogens.
• The assay can be effectively used for non-lethal amphibian health monitoring and disease surveillance.
• While Bsal was not found, continued vigilance is necessary given its high pathogenicity.