Acid-activated bentonite for solid-phase nucleic acid extraction from various pathogenic samples

Abstract

Background

Despite significant advancements in nucleic acid testing technologies, current nucleic acid extraction methods are often limited by inefficiency, complexity, and a lack of versatility. To overcome these challenges, we have developed an innovative solid-phase extraction (SPE) method employing sulfuric acid-activated bentonite (SAB) for extracting nucleic acids from various sample types.

Results

Activation with sulfuric acid expands the surface area of bentonite by 2.2 times, thereby enhancing its adsorption capacity and surface modification efficiency. To further improve extraction efficiency, we modified SAB through amine-functionalization using 3-aminopropyl(diethoxy)methylsilane (APDMS), resulting in the creation of APDMS-modified SAB (ASAB). This modification facilitates efficient nucleic acid binding via reversible interactions mediated by a homobifunctional imidoester (HI) reagent. Our ASAB-based SPE system offers a streamlined, universal protocol for isolating DNA, RNA, and miRNA from diverse samples, including clinical bodily fluids and culture media, in under 30 min. Moreover, the system effectively enriches low concentrations of negatively charged pathogens (down to 20 CFU/reaction) from large-volume samples (up to 50 mL) through a 30-min pre-enrichment step utilizing the positively charged ASAB-HI complex. Comparative testing with pooled human urine and plasma samples revealed up to a 3.95-fold increase in DNA recovery compared to commercial SPE kits. Additionally, the system demonstrated up to a 6.3-fold improvement in the isolation of unstable viral RNA from clinical nasopharyngeal swabs, as well as critical microRNA biomarkers.

Significance

The versatility and high efficiency of nucleic acid recovery with our ASAB-based SPE system indicate its potential to revolutionize traditional SPE methods, positioning it as a universal nucleic acid extraction platform for molecular biology research.