Wide Dynamic Range Multiplex Digital Clustered Regularly Interspaced Short Palindromic Repeat Chip for Rapid...

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biosensor Technology

Background:

• Accurate multiplex nucleic acid detection is vital for infectious disease diagnosis and pathogen identification.
• Current methods face challenges in speed, sensitivity, and quantification capabilities.

Purpose of the Study:

• To develop a wide dynamic range multiplex digital clustered regularly interspaced short palindromic repeat (CRISPR) chip (WDRM-dCRISPR).
• To establish a rapid, sensitive, multiplexed nucleic acid detection (RSMND) platform for absolute quantification.

Main Methods:

• Integration of recombinase polymerase amplification (RPA), CRISPR/Cas12a specific cleavage, and microfluidics.
• Development of a digital absolute quantification method for multiplex nucleic acids.

Main Results:

• The RSMND platform achieved a limit of detection of 90 copies/mL within 30 minutes.
• Demonstrated a linear dynamic range of 9 × 10^1 to 1.8 × 10^6 copies/mL (R^2 > 0.9999) for foodborne pathogens.
• Showcased high sensitivity, specificity, and tolerance to background interference, with successful application in milk analysis.

Conclusions:

• The WDRM-dCRISPR chip and RSMND platform provide precise and reliable digital absolute quantification of multiplex nucleic acids.
• The platform offers superior accuracy and sensitivity compared to quantitative polymerase chain reaction (qPCR).
• This technology holds significant potential for point-of-care testing applications in food safety and infectious disease diagnostics.