Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP.
Lisa BechererJacob Friedrich HessSieghard FrischmannMohammed BakheitHans NitschkoSilvina StincoFriedrich ZitzHannes HoferGiampiero PorroFlorian HausladenKarl StockDominik DrossartHolger WurmHanna KuhnDominik HuberTobias HutzenlaubNils PaustMark KellerOliver StrohmeierSimon WadleNadine BorstRoland ZengerleFelix von StettenPublished in: Micromachines (2021)
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.
Keyphrases
- nucleic acid
- loop mediated isothermal amplification
- high throughput
- induced apoptosis
- deep learning
- single cell
- machine learning
- sensitive detection
- cell cycle arrest
- cell therapy
- endothelial cells
- endoplasmic reticulum stress
- stem cells
- sars cov
- crispr cas
- high resolution
- oxidative stress
- signaling pathway
- cell proliferation
- copy number
- dna methylation
- pi k akt
- molecular dynamics
- circulating tumor
- real time pcr