A visual CRISPR/dCas9-mediated enzyme-linked immunosorbent assay for nucleic acid detection with single-base...

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Nucleic acid detection is vital for molecular diagnostics, especially in resource-limited settings.
• Existing detection methods often lack specificity.
• The clustered regularly interspaced short palindromic repeats (CRISPR) system offers precise DNA recognition.

Purpose of the Study:

• To develop a specific, sensitive, and economical nucleic acid detection method for genetically modified (GM) crops.
• To utilize the nuclease-dead Cas9 (dCas9)/sgRNA system for enhanced DNA recognition.
• To establish a visual clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated enzyme-linked immunosorbent assay (ELISA) for detecting the cauliflower mosaic virus 35s (CaMV35S) promoter.

Main Methods:

• Amplification of the CaMV35S promoter using biotinylated primers.
• Complex formation between the amplified DNA, dCas9, and sgRNA.
• Capture of the complex on an antibody-coated microplate.
• Detection using a streptavidin-labeled horseradish peroxidase probe for visual readout.

Main Results:

• The dCas9-ELISA method achieved a detection limit as low as 12.5 copies μL^-1 for the CaMV35S promoter.
• The assay demonstrated single-base specificity, distinguishing target sequences accurately.
• Combined with rapid amplification techniques, the method identified GM rice seeds within 1.5 hours.

Conclusions:

• The developed CRISPR/dCas9-ELISA is a specific, sensitive, rapid, and cost-effective platform for molecular diagnostics.
• This method is suitable for resource-limited settings, requiring no expensive equipment or specialized expertise.
• It offers a viable solution for identifying genetically modified organisms in crops.