Thin Film Microextraction Enables Rapid Isolation and Recovery of DNA for Downstream Amplification Assays.

ABSTRACT

Nucleic acid analysis has been at the forefront of the COVID-19 global health crisis where millions of diagnostic tests have been used to determine disease status as well as sequencing techniques that monitor the evolving genome of SARS-CoV-2. In this study, we report the development of a sample preparation method that decreases the time required for DNA isolation while significantly increasing the sensitivity of downstream analysis. Functionalized planar supports are modified with a polymeric ionic liquid sorbent coating to form thin film microextraction (TFME) devices. The extraction devices are shown to have a high affinity for DNA while also exhibiting high reproducibility and reusability. Using quantitative polymerase chain reaction (qPCR) analysis, the TFME devices are shown to require low equilibration times while achieving higher preconcentration factors than solid-phase microextraction (SPME) by extracting larger masses of DNA. Rapid desorption kinetics enable higher DNA recoveries using desorption solutions that are less inhibitory to qPCR and loop-mediated isothermal amplification (LAMP). To demonstrate the advantageous features of the TFME platform, a customized leuco crystal violet LAMP assay is used for visual detection of the ORF1ab DNA sequence from SARS-CoV-2 spiked into artificial oral fluid samples. When coupled to the TFME platform, 100% of LAMP reactions were positive for SARS-CoV-2 compared to 66.7% obtained by SPME for a clinically relevant concentration of 4.80 × 106 DNA copies/mL.