Nucleic acid extraction from complex biofluid using toothpick-actuated over-the-counter medical-grade cotton.

ABSTRACT

Nucleic acid amplification technique (NAAT)-assisted detection is the primary intervention for pathogen molecular diagnostics. However, NAATs such as quantitative real-time polymerase chain reaction (qPCR) require prior purification or extraction of target nucleic acid from the sample of interest since the latter often contains polymerase inhibitors. Similarly, genetic disease screening is also reliant on the successful extraction of pure patient genomic DNA from the clinical sample. However, such extraction techniques traditionally utilize spin-column techniques that in turn require centralized high-speed centrifuges. This hinders any potential deployment of qPCR- or PCR-like NAAT methods in resource-constrained settings. The development of instrument-free nucleic acid extraction methods, especially those utilizing readily available materials would be of great interest and benefit to NAAT-mediated molecular diagnosis workflows in resource-constrained settings. In this report, we screened medical-grade cotton, a readily available over-the-counter biomaterial to extract genomic DNA (gDNA) spiked in 30 %, 45 %, and 60 % serum or cell lysate. The extraction was carried out in a completely instrument-free manner using cotton and a sterilized toothpick and was completed in 30 min (with using chaotropic salt) or 10 min (without using chaotropic salt). The quality of the extracted DNA was then probed using PCR followed by agarose gel analysis for preliminary validation of the study. The qPCR experiments then quantitatively established the extraction efficiency (0.3-27 %, depending on serum composition). Besides, percent similarity score obtained from the Sanger sequencing experiments probed the feasibility of extracted DNA towards polymerase amplification with fluorescent nucleotide incorporation. Overall, our method demonstrated that DNA extraction could be performed utilizing toothpick-mounted cotton both with or without using a chaotropic salt, albeit with a difference in the quality of the extracted DNA.