An automated and portable platform for rapid cell-free DNA isolation and its application in microbial DNA metagenomic sequencing from human blood samples

TY - JOUR

T1 - An automated and portable platform for rapid cell-free DNA isolation and its application in microbial DNA metagenomic sequencing from human blood samples

AU - Marriott, Linda

AU - Martinez-Lopez, Ana

AU - Liga, Antonio

AU - Horiba, Kazuhiro

AU - Warr, Amanda

AU - Phulusa, Jacob N.

AU - Kumar, Radhe Shantha

AU - Carey, Laura

AU - Ito, Yoshinori

AU - Parcell, Benjamin J.

AU - Leslie, Nicholas R.

AU - Feasey, Nicholas A.

AU - Jacob, Shevin T.

AU - Rylance, Jamie

AU - Kersaudy-Kerhoas, Maïwenn

N1 - Publisher Copyright: This journal is © The Royal Society of Chemistry, 2026

PY - 2026/5/5

Y1 - 2026/5/5

N2 - 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, 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 and a consumable (CNASafe) for cfDNA isolation from human plasma samples. The platform-device performance was evaluated by comparing relative 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 1 hour 15 min 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.

AB - 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, 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 and a consumable (CNASafe) for cfDNA isolation from human plasma samples. The platform-device performance was evaluated by comparing relative 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 1 hour 15 min 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.

UR - https://www.scopus.com/pages/publications/105035243920

U2 - 10.1039/d5lc00876j

DO - 10.1039/d5lc00876j

M3 - Article

C2 - 41949263

AN - SCOPUS:105035243920

SN - 1473-0197

VL - 26

SP - 2849

EP - 2860

JO - Lab on a Chip

JF - Lab on a Chip

IS - 9

ER -