B-232 Versatile, Extraction-Free Nucleic Acid Sample Preparation in 15 Seconds Via Hyperbaric Heating

Abstract

Abstract Nucleic acid extraction-based PCR sample prep is currently limited by long processing times, liquid transfers, kit cost, and lab storage requirements. Extraction-free approaches enable streamlined sample prep, but lack sensitivity compared to extraction-based methods. We studied several sample types to demonstrate the effectiveness of a novel 15-second Hyperbaric Heating (HBH) process to prepare nucleic acids for PCR. HBH exposes the sample to a short duration, high temperature environment to effectively lyse organisms and denature PCR inhibitors such as nucleases in seconds. Samples are introduced and sealed in a pressure-tight metallic vessel, which is then heated in seconds using magnetic induction. Within the sealed metallic vessel, the vaporization of the aqueous sample raises the internal pressure, enabling an internal temperature higher than 100°C to be achieved. Chelating agents and other proteins in HBH also protect the target nucleic acids from fragmentation. Post HBH, the sample is ready for direct PCR amplification without further dilution or extraction. Efficacy of HBH for efficient PCR amplification was tested against commercial kits. HBH was applied to viral, bacterial, eukaryotic, and fungal organisms. For SARS-CoV-2, HBH detected down to 250 viral copies/mL. Additionally, HBH outperformed commercial kits for PCR sample prep of Candida albicans and Bacillus subtilis. HBH is a generalizable sample prep technology with sensitivity comparable to that of current extraction kits. We have applied HBH to a multitude of organisms and have demonstrated that HBH can be employed as a rapid and comprehensive nucleic acid prep approach for applications that involve PCR amplification post sample prep. Future integration of HBH in devices suitable for point of care molecular testing are underway. We also envision HBH to be a useful, generalizable sample prep method for the molecular lab in applications where cost, simplicity, and speed are essential for preparing nucleic acids for amplification.