| Literature DB >> 33562822 |
Lisa Becherer1,2, Jacob Friedrich Hess1,2, Sieghard Frischmann3, Mohammed Bakheit3, Hans Nitschko4, Silvina Stinco4, Friedrich Zitz5, Hannes Hofer5, Giampiero Porro6, Florian Hausladen7, Karl Stock7, Dominik Drossart7, Holger Wurm7, Hanna Kuhn1,8, Dominik Huber1,2, Tobias Hutzenlaub1,2, Nils Paust1,2, Mark Keller1,2,9, Oliver Strohmeier1,2,9, Simon Wadle1,2,10, Nadine Borst1,2, Roland Zengerle1,2, Felix von Stetten1,2.
Abstract
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.Entities:
Keywords: centrifugal microfluidics; digital loop-mediated isothermal amplification; human T-cell lymphotropic virus 1; point-of-care testing; proviral load measurements
Year: 2021 PMID： 33562822 PMCID： PMC7915047 DOI： 10.3390/mi12020159
Source DB: PubMed Journal: Micromachines (Basel) ISSN： 2072-666X Impact factor: 2.891