Point-of-care system for htlv-1 proviral load quantification by digital mediator displacement lamp

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.