LabDisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza A H3N2 virus

• Published in: Lab on a Chip 2016-01, Vol.16 (1), p.199-207
• Main Authors: Stumpf, F, Schwemmer, F, Hutzenlaub, T, Baumann, D, Strohmeier, O, Dingemanns, G, Simons, G, Sager, C, Plobner, L, von Stetten, F, Zengerle, R, Mark, D
• Format: Article
• Language: English
• Subjects: Devices; Extraction; Indicators and Reagents - chemistry; Influenza; Influenza A Virus, H3N2 Subtype - genetics; Influenza A Virus, H3N2 Subtype - isolation & purification; Microfluidic Analytical Techniques - instrumentation; Microfluidics; Nucleic acids; Pathogens; Point-of-Care Systems; Reagents; Real-Time Polymerase Chain Reaction - instrumentation; RNA, Viral - analysis; RNA, Viral - genetics; Viruses
• ISSN: 1473-0197; 1473-0189
• DOI: 10.1039/c5lc00871a

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.