SHERLOCK Assay for Rapid Detection of Batrachochytrium dendrobatidis

How SHERLOCK can help amphibians

Objective

Develop a rapid, sensitive, and field-deployable CRISPR-based SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) assay to detect B. dendrobatidis.

Project Significance

Once developed, the chytrid SHERLOCK assay will assist in disease mitigation strategies by producing on-site answers concerning infection status of in-hand amphibians or water bodies.

Project Summary

Chytridiomycosis, or chytrid, is an infectious amphibian disease caused by the that was spread by the global amphibian trade to all continents except Antarctica. Chytrid has been detected in 55% of amphibian species and implicated in the decline of 501 species and extinction in the wild for up to 90 species. Chytrid is caused by the aquatic fungus Batrachochytrium dendrobatidis (Bd), which resides in water as zoospores. These spores adhere to amphibian skin,

where they encyst and form a spore case that produces and expels additional spores into the environment. Chytrid mitigation strategies rely on the rapid detection of small quantities of Bd, but quick diagnosis is currently a major challenge.

Currently, scientists screen for Bd using quantitative polymerase chain reaction (qPCR) which amplifies and detects zoospore DNA on skin swabs or in water samples. Although this technique is sensitive and specific, a major drawback is that sample processing must occur in a lab using expensive equipment, increasing cost and introducing a potentially significant time gap between sample collection and pathogen detection. SHERLOCK assays use the CRISPR enzyme

Cas13a to target a specific genomic sequence within an environmental sample (e.g., skin swab, water). If the target DNA sequence is detected, the enzyme initiates a chain reaction that

produces a signal on an inexpensive handheld fluorescence reader within 30 minutes. Previously developed SHERLOCK assays can detect minute quantities of target DNA and exhibit less variation than qPCR. Additionally, SHERLOCK assays require minimal formal training to use and are inexpensive to produce once developed.