期刊
LAB ON A CHIP
卷 15, 期 14, 页码 3065-3075出版社
ROYAL SOC CHEMISTRY
DOI: 10.1039/c5lc00462d
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- National Institute for Health Research (NIHR), Rapid detection of infectious agents at point of triage (PoT) [II-ES-0511-21002]
- National Institute for Health Research [II-ES-0511-21002] Funding Source: researchfish
The widespread dissemination of CTX-M extended spectrum beta-lactamases among Escherichia coli bacteria, both in nosocomial and community environments, is a challenge for diagnostic bacteriology laboratories. We describe a rapid and sensitive detection system for analysis of DNA containing the bla(CTX-M-15) gene using isothermal DNA amplification by recombinase polymerase amplification (RPA) on a digital microfluidic platform; active matrix electrowetting-on-dielectric (AM-EWOD). The devices have 16800 electrodes that can be independently controlled to perform multiple and simultaneous droplet operations. The device includes an in-built impedance sensor for real time droplet position and size detection, an on-chip thermistor for temperature sensing and an integrated heater for regulating the droplet temperature. Automatic dispensing of droplets (45 nL) from reservoir electrodes is demonstrated with a coefficient of variation (CV) in volume of approximately 2%. The RPA reaction is monitored in real-time using exonuclease fluorescent probes. Continuous mixing of droplets during DNA amplification significantly improves target DNA detection by at least 100 times compared to a benchtop assay, enabling the detection of target DNA over four-order-of-magnitude with a limit of detection of a single copy within similar to 15 minutes.
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