In-Field Molecular Diagnostics of Plant Pathogens Using Bioluminescent CRISPR-Guided Caspase Assay.

Area of Science:

• Plant Pathology
• Molecular Diagnostics
• Biotechnology

Background:

• In-field molecular diagnostics are crucial for effective crop disease management and precision agriculture.
• Current diagnostic tools often lack the necessary sensitivity, speed, or field deployability.
• Detecting viable pathogens is essential to avoid misdiagnosis and ensure accurate disease risk assessment.

Purpose of the Study:

• To develop a novel, field-deployable molecular diagnostic assay for detecting viable plant pathogens.
• To enable rapid, sensitive, and precise in-field diagnosis of crop diseases.
• To overcome limitations of existing methods, such as PCR, by specifically detecting viable pathogens.

Main Methods:

• Development of bioluminescent craspase diagnostics (BioCrastics) using a novel RNA-activated protease of CRISPR (Craspase).
• Utilizing enzymatic luminescence and cascaded amplification for high sensitivity without nucleic acid purification or amplification.
• Demonstration using wheat stripe rust, analyzing crude leaf homogenates.

Main Results:

• Achieved sub-nanogram sensitivity for fungal pathogens.
• Enabled direct pathogen detection in crude plant samples within 40 minutes.
• Identified infections 6 days before visible symptoms emerged.
• Specifically quantified viable fungi, avoiding false positives from dead pathogens.

Conclusions:

• BioCrastics offers a field-deployable solution for rapid and precise crop disease diagnosis.
• The assay's ability to detect viable pathogens addresses a key limitation of current molecular methods.
• This technology bridges the gap between laboratory diagnostics and on-farm needs for improved crop management.