Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis.

Area of Science:

• Molecular Biology
• Biotechnology
• Point-of-Care Diagnostics

Background:

• Quantitative nucleic acid amplification tests (qNAATs) are vital for infectious disease management, including HIV viral load monitoring and SARS-CoV-2 testing.
• Current gold standard quantitative PCR (qPCR) requires laboratory infrastructure, limiting its use in resource-limited settings or at the point-of-care (POC).
• Isothermal amplification methods offer a promising alternative for developing POC NAATs, but accurate quantification remains a challenge.

Conclusions:

• The number of fluorescent nucleation sites in RPA is a reliable metric for nucleic acid quantification.
• This method enables rapid, sensitive, and accurate qNAATs at the point-of-care.
• The mobile phone-integrated system offers a low-cost, accessible solution for infectious disease diagnostics in diverse settings.