Optical-Controlled One-Pot RPA-CRISPR Assay for Environmental DNA Detection of a Critically Endangered Species

Area of Science:

• Molecular Biology
• Environmental Science
• Conservation Genetics

Background:

• Current polymerase chain reaction (PCR)-based assays for environmental DNA (eDNA) detection are slow, expensive, and require laboratory thermal cycling.
• Rapid, sensitive, and field-deployable assays are crucial for on-site monitoring of endangered species.

Purpose of the Study:

• To adapt and validate a previously reported optically controlled one-pot RPA-CRISPR-Cas12a (OORC) assay for the detection of eDNA from the critically endangered Bahaba taipingensis.
• To develop a portable and sensitive workflow for on-site eDNA monitoring.

Main Methods:

• Combined isothermal recombinase polymerase amplification (RPA) with CRISPR-Cas12a trans-cleavage in a single tube (OORC assay).
• Operated the entire process at a constant temperature, eliminating the need for thermal cycling.
• Integrated the OORC assay with a portable handheld fluorescence detector for photoactivation and fluorescence readout.

Main Results:

• The OORC assay demonstrated high specificity and sensitivity, with a redefined limit of detection of 6 copies per reaction.
• The integrated portable system enabled rapid, on-site detection of Bahaba taipingensis eDNA.
• The assay successfully operated at a constant temperature, simplifying field deployment.

Conclusions:

• The adapted OORC assay provides a rapid, sensitive, and field-deployable method for eDNA detection of endangered species.
• This portable workflow enhances the potential of eDNA monitoring for conservation applications.
• The assay offers a practical tool for the effective conservation of critically endangered species like Bahaba taipingensis.