Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP

• Main Authors: Lisa Becherer, Jacob Friedrich Hess, Sieghard Frischmann, Mohammed Bakheit, Hans Nitschko, Silvina Stinco, Friedrich Zitz, Hannes Hofer, Giampiero Porro, Florian Hausladen, Karl Stock, Dominik Drossart, Holger Wurm, Hanna Kuhn, Dominik Huber, Tobias Hutzenlaub, Nils Paust, Mark Keller, Oliver Strohmeier, Simon Wadle, Nadine Borst, Roland Zengerle, Felix von Stetten
• Format: Article
• Language: English
• Published: MDPI AG 2021-02-01
• Series: Micromachines
• Subjects: human T-cell lymphotropic virus 1; digital loop-mediated isothermal amplification; point-of-care testing; centrifugal microfluidics; proviral load measurements
• Online Access: https://www.mdpi.com/2072-666X/12/2/159
• ISSN: 2072-666X

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.