A digital microfluidic system for loop-mediated isothermal amplification and sequence specific pathogen detection

Liang Wan,Jie Gao,Chu-Xia Deng,Cheng Dong,Rui P Martins,Ada Hang-Heng Wong,Tianlan Chen,Pui-In Mak,Yanwei Jia

doi:10.1038/s41598-017-14698-x

Abstract

A digital microfluidic (DMF) system has been developed for loop-mediated isothermal amplification (LAMP)-based pathogen nucleic acid detection using specific low melting temperature (Tm) Molecular Beacon DNA probes. A positive-temperature-coefficient heater with a temperature sensor for real-time thermal regulation was integrated into the control unit, which generated actuation signals for droplet manipulation. To enhance the specificity of the LAMP reaction, low-Tm Molecular Beacon probes were designed within the single-stranded loop structures on the LAMP reaction products. In the experiments, only 1 μL of LAMP reaction samples containing purified Trypanosoma brucei DNA were required, which represented over a 10x reduction of reagent consumption when comparing with the conventional off-chip LAMP. On-chip LAMP for unknown sample detection could be accomplished in 40 min with a detection limit of 10 copies/reaction. Also, we accomplished an on-chip melting curve analysis of the Molecular Beacon probe from 30 to 75 °C within 5 min, which was 3x faster than using a commercial qPCR machine. Discrimination of non-specific amplification and lower risk of aerosol contamination for on-chip LAMP also highlight the potential utilization of this system in clinical applications. The entire platform is open for further integration with sample preparation and fluorescence detection towards a total-micro-analysis system.

Highlights

Digital microfluidics (DMF) is an emerging technology in the microfluidic field to manipulate individual microliter- to nanoliter-sized droplets on an array of electrodes by electro-wetting force[1]
Efforts have been made to discriminate non-specific amplification using nucleic acid probes that targeted specific single-stranded sequences in the loop structures of the Loop-mediated isothermal amplification (LAMP) products, such as biotinylated probes used in southern blotting[21], oligo DNA probes with cationic polymers enabling naked-eyed visualization[22], FITC-labeled probes utilized in lateral flow dipstick[23], alternative binding quenching probes[24], and fluorescence energy transfer-based probes[25]
Other copies of repetitive insertion mobile element (RIME) with nucleotide variations in the primer and probe binding sites have been found in the T. brucei genome and have a chance to be detected

Summary

Introduction

Digital microfluidics (DMF) is an emerging technology in the microfluidic field to manipulate individual microliter- to nanoliter-sized droplets on an array of electrodes by electro-wetting force[1]. Efforts have been made to discriminate non-specific amplification using nucleic acid probes that targeted specific single-stranded sequences in the loop structures of the LAMP products, such as biotinylated probes used in southern blotting[21], oligo DNA probes with cationic polymers enabling naked-eyed visualization[22], FITC-labeled probes utilized in lateral flow dipstick[23], alternative binding quenching probes[24], and fluorescence energy transfer-based probes[25]. These additional technologies either require post-amplification manipulation, which may lead to cross contamination, or involve complicated probe designs and assay settings. This protocol, lowers reaction efficiency since the Molecular Beacon probes has to be displaced during DNA amplification

Methods

Results

Conclusion