基于数字介导置换环介导等温扩增技术的人嗜T淋巴细胞病毒1型前病毒载量即时检测系统

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

作者信息

Becherer Lisa, Hess Jacob Friedrich, Frischmann Sieghard, Bakheit Mohammed, Nitschko Hans, Stinco Silvina, Zitz Friedrich, Hofer Hannes, Porro Giampiero, Hausladen Florian, Stock Karl, Drossart Dominik, Wurm Holger, Kuhn Hanna, Huber Dominik, Hutzenlaub Tobias, Paust Nils, Keller Mark, Strohmeier Oliver, Wadle Simon, Borst Nadine, Zengerle Roland, von Stetten Felix

机构信息

Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.

Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.

出版信息

Micromachines (Basel). 2021 Feb 5;12(2):159. doi: 10.3390/mi12020159.

DOI:10.3390/mi12020159

PMID:33562822

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7915047/

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.

摘要

本文介绍了一种基于数字逆转录RNA和MD LAMP（介导置换环介导的等温扩增）的cDNA扩增技术，用于全自动定量检测人1型嗜T细胞病毒（HTLV-1）前病毒载量（包括基因组RNA）的通用即时检测系统。一种预存试剂的一次性微流控实验盘可实现自动核酸提取、反应设置、乳化、逆转录、数字DNA扩增以及使用通用报告分子进行定量荧光终点检测。从HTLV-1感染的CD4+ T淋巴细胞（MT-2细胞）悬液中自动提取核酸，每个MT-2细胞产生8±7个病毒核酸拷贝，与手动参考提取法（7±2个核酸拷贝）非常相似。对添加MT-2细胞的全血进行全自动样本处理，在运行2小时10分钟后，每个MT-2细胞的检测结果为7±3个拷贝，结果相当。