StratoLAMP: Label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of precipitate

Abstract

Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness.

Rapid Detection of Uropathogens Using an Integrated Multiplex Digital Nucleic Acid Detection Assay Powered by a Digital-to-Droplet Microfluidic Device

“…Consequently, droplet digital assays are highly advantageous for rapid and precise pathogen detection. ,, Currently, droplet digital analysis has been adapted to various types of biomedical detection assays. A variety of digital nucleic acid detection methods have been developed recently. − However, these multiplexed assays involve a series of procedures, including nucleic acid extraction, droplet generation, nucleic acid amplification, and droplet detection, which are typically performed offline . Consequently, these assays necessitate a specialized laboratory and a considerable turnaround time.…”