Rapid automatic nucleic acid purification system based on gas-liquid immiscible phase.

The continuous expansion of nucleic acid detection applications has resulted in constant developments in rapid, low-consumption, and highly automated nucleic acid extraction methods. Nucleic acid extraction using magnetic beads across an immiscible phase interface offers significant simplification and parallelization potential. The gas-liquid immiscible phase valve eliminates the requirement for complicated cassettes and is suitable for automation applications. By analyzing the process of magnetic beads crossing the gas-liquid interface, we utilized a low magnetic field strength to drive large magnetic bead packages to cross the gas-liquid interface, providing a solution of high magnetic bead recovery rate for solid-phase extraction with a low-surfactant system based on gas-liquid immiscible phase valve. The recovery rate of magnetic beads was further improved to 90%-95% and the carryover of the reagents was below 1%. Consequently, a chip and an automatic system were developed to verify the applicability of this method for nucleic acid extraction. The Hepatitis B virus serum standard was used for the extraction test. The extraction of four samples was performed within 7 minutes, with nucleic acid recovery maintained above 80% and good purity. Thus, through analysis and experiments, a fast, highly automated, and low-consumption nucleic acid recovery method was proposed in this study.

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

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.

Comparison of Rapid Nucleic Acid Extraction Methods for SARS-CoV-2 Detection by RT-qPCR.

Since 2020, humanity has been facing the COVID-19 pandemic, a respiratory disease caused by the SARS-CoV-2. The world's response to pandemic went through the development of diagnostics, vaccines and medicines. Regarding diagnostics, an enormous challenge was faced due to shortage of materials to collect and process the samples, and to perform reliable mass diagnosis by RT-qPCR. In particular, time-consuming and high cost of nucleic acid extraction procedures have hampered the diagnosis; moreover, several steps in the routine for the preparation of the material makes the extracted sample susceptible to contamination. Here two rapid nucleic acid extraction reagents were compared as extraction procedures for SARS-CoV-2 detection in clinical samples by singleplex and multiplex RT-qPCR analysis, using different transport media, samples with high and low viral load, and different PCR machines. As observed, rapid nucleic acid extraction procedures can be applied for reliable diagnosis using a TaqMan-based assay, over multiple platforms. Ultimately, prompt RNA extraction may reduce costs with reagents and plastics, the chances of contamination, and the overall time to diagnosis by RT-qPCR.