An Automated and Portable Platform for Rapid Cell-Free DNA Isolation and Its Application in Microbial DNA metagenomic Sequencing from Human Blood Samples

Abstract

The prompt identification of pathogens in human circulation in a clinically deployable format remains an unmet clinical need. The established test for infection diagnostics remains blood culture, which typically takes 2-4 days and is positive in less than 15% of cases, with many prevalent pathogens difficult or impossible to culture. While microbial cfDNA in blood could facilitate the diagnosis of sepsis and febrile and infectious conditions, sample preparation for cell-free DNA (cfDNA) analysis in decentralised settings presents challenges due to its complexity and the low concentration and fragmented nature of cfDNA in blood plasma. We developed a portable and automated platform (CNASafe) for cfDNA isolation from human plasma samples. Device performance was evaluated by comparing cfDNA yield against a reference (QIAGEN QIAamp Circulating Nucleic Acid Kit). cfDNA eluates from ten non-cultured blood samples from hospital patients were sequenced on a nanopore sequencer, and results compared to blood cultures. Extraction of cfDNA using the CNASafe device was completed in 40 minutes, compared to the 2-hour reference protocol. The device achieved an average relative cfDNA recovery of 100.5% over 333 unique extractions encompassing all parameter variations, demonstrating a performance equivalent to the reference kit. From the patient samples, a sufficient quantity of microbial cfDNA was extracted to either identify pathogens missed by blood cultures or confirm negative cultures. The CNASafe platform and real-time nanopore sequencing offer a promising solution for the rapid deployment of metagenomic diagnostics, enabling pathogen identification within a few hours in decentralised clinical environments.