B-216 Ethanol/isopropanol-free and enzyme-free universal nucleic acid extraction and purification system for fast molecular detection from swab samples
Authors: Zhang W , Boey E , Krishnamurth C , Ng L 
Published: October 2024

Abstract

Background
Efficient nucleic acid (NA) extraction has been made technology advancement in molecular science. However, there are several challenges yet to address. Firstly, ethanol or isopropanol are important components of the buffers for the most commercially available NA extraction kits. When put on cartridge for POC applications, they can react with plastic materials, undermining their mechanical/physiochemical properties. They can also be problematic due to their volatility, flammability and potential to leak. Secondly, turn-around time for most commercial kits is long and procedure is rather tedious. Thirdly, most of the extraction buffers can only be used to extract either DNA or RNA from either viruses or bacteria. A universal buffer system is lacking. Lastly, special components in some extraction buffers require -20°C or 4°C storage for desired performance, especially for those kits containing proteinase for cell lysis and nuclease denaturation and carrier NA for low quantity of template, causing inconvenience in shipping and storage. Delta developed buffers and associated protocol to address the challenges listed above. This study is to evaluate the performance of the Delta TNA buffer system in NA extraction and purification from various target pathogens in swab samples when compared to that of the leading commercial products.

Methods
The stock strains of staphylococcus aureus, pseudomonas aeruginosa and H-coronavirus OC43 were diluted in Delta Molecular Transport Media containing swab sample matrix to the testing concentrations before performing NA extraction. Vircell SARS-CoV-2 RNA was spiked directly into PCR reactions to generate a standard curve with 10x serial dilutions for the recovery rate test using Delta TNA kit. 300ul of samples were used for the NA extraction and purification with 80 ul eluate generated. The manufacturers’ instructions were followed. The extracted RNA or DNA was amplified on BioRAD CFX96.

Results
The extraction efficiency on RNA from H-coronavirus OC43, DNA from pseudomonas aeruginosa and staphylococcus aureus in swab samples, was compared with Katsura Respi and MN Pathogen kits, Qiagen DNA and viral RNA kits respectively. Delta TNA kit outperformed in either earlier Cq value or higher detection rate for different concentrations of analytes in &lt;10 min comparing to up to 95 min for the commercial kits tested. The RNA recovery rate of Delta TNA kit was comparable to that of the Qiagen Viral Kit with 37.54% vs 29.84% at 10 copies/rxn, 146.84% vs 140.76% at 100 cpies/rxn and 118.29% vs 111.76% at 1000 copies/rxn. Meanwhile, Delta Swab TNA buffer was compatible with large spectrum of magnetic beads possessing various coating groups and particle sizes.

Conclusions
We provided a stable, user- and environment-friendly total nucleic acid extraction and purification system which is ethanol/isopropanol-free and enzyme-free. There is no pre-treatment or drying steps in the protocol and no centrifugation required. Delta’s protocol has the least total number of steps and least amount of total time taken amongst the kits compared. It serves as a universal recipe for both DNA and RNA from pathogens in swab samples including viruses and bacteria in less than 10 min, with competitive performance in both PCR and LAMP.