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Issue 24, 2021
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Multiplexed droplet loop-mediated isothermal amplification with scorpion-shaped probes and fluorescence microscopic counting for digital quantification of virus RNAs

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Abstract

Highly sensitive digital nucleic acid techniques are of great significance for the prevention and control of epidemic diseases. Here we report the development of multiplexed droplet loop-mediated isothermal amplification (multiplexed dLAMP) with scorpion-shaped probes (SPs) and fluorescence microscopic counting for simultaneous quantification of multiple targets. A set of target-specific fluorescence-activable SPs are designed, which allows establishment of a novel multiplexed LAMP strategy for simultaneous detection of multiple cDNA targets. The digital multiplexed LAMP assay is thus developed by implementing the LAMP reaction using a droplet microfluidic chip coupled to a droplet counting microwell chip. The droplet counting system allows rapid and accurate counting of the numbers of total droplets and the positive droplets by collecting multi-color fluorescence images of the droplets in a microwell. The multiplexed dLAMP assay was successfully demonstrated for the quantification of HCV and HIV cDNA with high precision and detection limits as low as 4 copies per reaction. We also verified its potential for simultaneous digital assay of HCV and HIV RNA in clinical plasma samples. This multiplexed dLAMP technique can afford a useful platform for highly sensitive and specific detection of nucleic acids of viruses and other pathogens, enabling rapid diagnosis and prevention of infectious diseases.

Graphical abstract: Multiplexed droplet loop-mediated isothermal amplification with scorpion-shaped probes and fluorescence microscopic counting for digital quantification of virus RNAs

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Supplementary files

Article information


Submitted
01 Feb 2021
Accepted
14 May 2021
First published
14 May 2021

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2021,12, 8445-8451
Article type
Edge Article

Multiplexed droplet loop-mediated isothermal amplification with scorpion-shaped probes and fluorescence microscopic counting for digital quantification of virus RNAs

Y. Tan, A. Huang, L. Tang and J. Jiang, Chem. Sci., 2021, 12, 8445
DOI: 10.1039/D1SC00616A

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    [Original citation] - Published by The Royal Society of Chemistry.

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