title: LabDisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza A H3N2 virus.
abstract: Portable point-of-care devices for pathogen detection require easy, minimal and user-friendly handling steps and need to have the same diagnostic performance compared to centralized laboratories. In this work we present a fully automated sample-to-answer detection of influenza A H3N2 virus in a centrifugal LabDisk with complete prestorage of reagents. Thus, the initial supply of the sample remains the only manual handling step. The self-contained LabDisk automates by centrifugal microfluidics all necessary process chains for PCR-based pathogen detection: pathogen lysis, magnetic bead based nucleic acid extraction, aliquoting of the eluate into 8 reaction cavities, and real-time reverse transcription polymerase chain reaction (RT-PCR). Prestored reagents comprise air dried specific primers and fluorescence probes, lyophilized RT-PCR mastermix and stick-packaged liquid reagents for nucleic acid extraction. Employing two different release frequencies for the stick-packaged liquid reagents enables on-demand release of highly wetting extraction buffers, such as sequential release of lysis and binding buffer. Microfluidic process-flow was successful in 54 out of 55 tested LabDisks. We demonstrate successful detection of the respiratory pathogen influenza A H3N2 virus in a total of 18 LabDisks with sample concentrations down to 2.39 × 10(4) viral RNA copies per ml, which is in the range of clinical relevance. Furthermore, we detected RNA bacteriophage MS2 acting as internal control in 3 LabDisks with a sample concentration down to 75 plaque forming units (pfu) per ml. All experiments were applied in a 2 kg portable, laptop controlled point-of-care device. The turnaround time of the complete analysis from sample-to-answer was less than 3.5 hours.
authors.affiliation: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Fabian.Stumpf@Hahn-Schickard.de.
authors.forename: F
authors.initials: F
authors.lastname: Stumpf
authors.forename: F
authors.initials: F
authors.lastname: Schwemmer
authors.forename: T
authors.initials: T
authors.lastname: Hutzenlaub
authors.forename: D
authors.initials: D
authors.lastname: Baumann
authors.forename: O
authors.initials: O
authors.lastname: Strohmeier
authors.forename: G
authors.initials: G
authors.lastname: Dingemanns
authors.forename: G
authors.initials: G
authors.lastname: Simons
authors.forename: C
authors.initials: C
authors.lastname: Sager
authors.forename: L
authors.initials: L
authors.lastname: Plobner
authors.forename: F
authors.initials: F
authors.lastname: von Stetten
authors.forename: R
authors.initials: R
authors.lastname: Zengerle
authors.forename: D
authors.initials: D
authors.lastname: Mark
mesh_terms: D007202:Indicators and Reagents / Q000737:chemistry*; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics* / Q000302:isolation & purification*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D019095:Point-of-Care Systems; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation
chemical_list: D007202:Indicators and Reagents; D012367:RNA, Viral