One-Pot CRISPR/Cas12a Assay Based on Ultrashort HDA for Ultrasensitive and Universal Nucleic Acid Detection

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• Isothermal amplification techniques like helicase-dependent amplification (HDA) combined with CRISPR are advanced methods for nucleic acid detection.
• Existing methods may require optimization for speed and sensitivity.

Purpose of the Study:

• To develop a novel ultrashort mesophilic HDA (usHDA) for rapid nucleic acid amplification at 37 °C.
• To construct a one-pot usHDA-CRISPR/Cas12 assay for highly sensitive and specific nucleic acid detection.

Main Methods:

• Developed ultrashort mesophilic HDA (usHDA) for amplifying ultrashort sequences (~40 nt) at 37 °C within 30 minutes.
• Constructed a one-pot usHDA-CRISPR/Cas12a assay for nucleic acid detection.
• Validated the assay on clinical specimens for respiratory pathogens and Staphylococcus infections.

Main Results:

• The usHDA-CRISPR/Cas12a assay achieved a limit of detection of 5 aM and completed detection within 1 hour.
• Tested on 58 clinical specimens, the assay demonstrated 100% sensitivity and 100% specificity for influenza A virus detection compared to PCR.
• Identified 24 Staphylococcus infection samples with 100% sensitivity and specificity.

Conclusions:

• The developed usHDA-CRISPR/Cas12a assay offers rapid, highly sensitive, and specific nucleic acid detection at a low temperature (37 °C).
• This assay shows strong applicability for universal nucleic acid detection in clinical settings.