Method for lysis and paper-based elution-free DNA extraction with colourimetric isothermal amplification

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Diagnostics

Background:

• Nucleic acid amplification testing offers high sensitivity and specificity for point-of-need diagnostics.
• Current sample preparation methods are complex, time-consuming, and require specialized equipment, hindering point-of-need applications.

Purpose of the Study:

• To develop a simplified, rapid sample preparation method for nucleic acid amplification testing.
• To integrate DNA extraction and purification into a single, user-friendly paper-based platform.

Main Methods:

• Utilized mixed cellulose ester (MCE) paper for DNA binding via ionic interaction under molecular crowding conditions.
• Employed a poly(ethylene) glycol (PEG)-based reagent for alkaline lysis and DNA binding.
• Introduced Paper-based Abridged Solid-Phase Extraction with Alkaline Poly(ethylene) Glycol Lysis (PASAP) for wicking-based fluid transport and DNA concentration.

Main Results:

• PASAP achieved DNA binding to MCE paper, concentrating DNA while matrix components wicked away.
• Sample processing, including washing and drying, was completed in 15 minutes.
• Colorimetric loop-mediated isothermal amplification (cLAMP) successfully detected *Escherichia coli* (E. coli) down to 10^2 CFU/mL in culture media and <10^3 CFU/mL in milk.

Conclusions:

• PASAP offers a streamlined, efficient, and instrument-free sample preparation method for nucleic acid testing.
• The paper-based approach simplifies workflows, eliminates the need for pipetting and magnetic beads.
• Demonstrated applicability in complex biological samples, paving the way for accessible point-of-need diagnostics.