A digital microfluidic system for loop-mediated isothermal amplification and sequence specific pathogen detection. Issue 1 (December 2017)

• Record Type: Journal Article
• Authors: Wan, Liang; Chen, Tianlan; Gao, Jie; Dong, Cheng; Wong, Ada; Jia, Yanwei; Mak, Pui-In; Deng, Chu-Xia; Martins, Rui
• Is Part Of: Scientific reports. Volume 7:Issue 1(2017)
• Page Start: 1
• Page End: 11
• Publication Date: 2017-12
• Subjects: Natural history -- Research -- Periodicals; Biology -- Research -- Periodicals; Physical sciences -- Research -- Periodicals; Earth sciences -- Research -- Periodicals; Environmental sciences -- Research -- Periodicals; 502.85
• DOI: 10.1038/s41598-017-14698-x
• Languages: English
• ISSNs: 2045-2322

Abstract

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 purifiedTrypanosoma 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.