RPA-CRISPR/Cas12a-coupled microfluidic biosensor enabling on-site, sensitive quantification of Vibrio...

Area of Science:

• Biotechnology
• Food Safety
• Aquaculture Health

Background:

• Vibrio parahaemolyticus is a significant pathogen causing seafood-borne illness and aquatic animal diseases.
• Accurate, on-site detection is crucial for managing public health risks and aquaculture.
• Existing methods like qPCR are limited in field applications due to complex instrumentation.

Purpose of the Study:

• To develop a robust, field-deployable quantitative isothermal assay for Vibrio parahaemolyticus detection.
• To enable standard-curve-based quantification with in-run calibration and variance control.
• To establish a transferable methodology for quantitative RPA-CRISPR-based assays.

Main Methods:

• A centrifugal microfluidic system integrating reaction-volume locking and simultaneous standard-curve generation.
• A one-pot Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a assay with optimized crRNAs and reaction conditions.
• Concurrent analysis of standards and samples for reduced variability.

Main Results:

• Achieved a detection limit of 6.08 copies/μL and a linear quantitative range of 10^0-10^4 copies/μL (R² > 0.96).
• Demonstrated performance comparable to qPCR (AUC = 0.984) with acceptable intra- and inter-assay variability (RSD 2.63-6.07%).
• Validated on spiked and real seafood samples, showing reliable on-site quantification at approximately $3.30 per test.

Conclusions:

• The proposed methodological framework enables reliable quantitative detection using isothermal amplification under field conditions.
• The integrated microfluidic platform and RPA-CRISPR/Cas12a assay offer a low-cost, accurate solution for pathogen detection.
• This work advances field-deployable diagnostics for aquaculture and food safety, addressing critical public health concerns.