Method for colorimetric detection of double-stranded nucleic acid using leuco triphenylmethane dyes

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Loop-mediated isothermal amplification (LAMP) is a powerful DNA amplification technique for genetic testing.
• Current LAMP detection methods (turbidity, metallochromic indicators) have limitations.
• A simple, sensitive, and selective detection method for LAMP is needed.

Purpose of the Study:

• To develop and validate a novel colorimetric method for detecting LAMP products using leuco crystal violet (LCV).
• To assess the sensitivity, selectivity, and applicability of the LCV-based LAMP assay.

Main Methods:

• Leuco crystal violet (LCV), a derivative of crystal violet (CV), was employed as a colorimetric indicator.
• LCV is converted to CV in the presence of double-stranded DNA (dsDNA), the product of LAMP.
• The color change from colorless to violet indicates a positive LAMP reaction.

Main Results:

• The LCV assay demonstrated high sensitivity, with a detection limit of 7.1 ng/μl for dsDNA.
• The method showed high selectivity for dsDNA, with no interference from single-stranded DNA or dNTPs.
• LCV allows direct detection of LAMP products, enabling its use under diverse reaction conditions, including with pyrophosphatase.

Conclusions:

• Leuco crystal violet provides a simple, sensitive, and selective colorimetric method for detecting LAMP products.
• This LCV-based assay is suitable for point-of-care genetic testing due to its ease of use and visible readout.
• The method overcomes limitations of existing LAMP detection techniques, enhancing its diagnostic utility.