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A digital microfluidic system for loop-mediated isothermal amplification and sequence specific pathogen detection
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DOI：10.1038/s41598-017-14698-x.png)
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摘要
En 中文
A digital microfluidic (DMF) system has been developed for loop-mediated isothermal amplification (LAMP)-based pathogen nucleic acid detection using specific low melting temperature (T-m ) 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-T-m Molecular Beacon probes were designed within the single-stranded loop structures on the LAMP reaction products. In the experiments, only 1 mu L 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 degrees 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.
Keyword:
TRYPANOSOMA-BRUCEI
MOLECULAR BEACONS
VISUAL DETECTION
PLATFORM
LAMP
PCR
SENSITIVITY
POINT
ASSAY
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期刊
IF:
3.9
论文数:
28.0W
被引数:
83.5W
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引用论文
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