Colorimetric PCR Based on Pyrophosphate Displacement

Area of Science:

• Molecular Biology
• Biochemistry
• Diagnostics

Background:

• Quantitative PCR (qPCR) is the standard for molecular diagnostics.
• Colorimetric readouts offer simplified, decentralized PCR testing.
• Existing colorimetric PCR methods target dsDNA amplicons, not PCR byproducts.

Purpose of the Study:

• To develop a novel colorimetric PCR assay targeting pyrophosphate (Py), an inherent PCR product.
• To overcome challenges of Py detection in the presence of Mg2+ ions.
• To establish a simple, low-cost, and sensitive method for decentralized nucleic acid testing.

Main Methods:

• Introduced a Scandium(III)-hydroxynaphthol blue (Sc-HNB) complex for Py detection.
• Utilized the higher stability of Sc-HNB and Sc-Py complexes over Mg-HNB and Mg-Py.
• Demonstrated Py displacement of HNB from Sc-HNB in a Mg2+-independent manner for color change.

Main Results:

• Achieved Py-based colorimetric PCR with distinct color change.
• Demonstrated sensitivity comparable to conventional qPCR for target gene detection.
• Validated the method for human papillomavirus (HPV) detection in vaginal swabs, consistent with qPCR results.

Conclusions:

• The proposed Py-displacement colorimetric PCR is a simple, low-cost, and sensitive molecular diagnostic tool.
• The method is robust, compatible with existing PCR kits, and functional with basic heating (water bath).
• This assay shows promise for decentralized nucleic acid testing applications.