Journal
ANALYTICAL BIOCHEMISTRY
Volume 544, Issue -, Pages 98-107Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2017.12.031
Keywords
Drug-resistant Mycobacterium tuberculosis; Infectious disease diagnostics; Isothermal amplification; Lab-on-a-disc; Loop-mediated isothermal amplification; Centrifugal microfluidics; Point-of-care testing; Recombinase polymerase amplification
Funding
- ITF [ITS/227/12]
- CRF [CUHK1/CRF/12G]
- AoE from the Hong Kong Special Administrative Region, VC Discretionary Fund [AoE/P-0/12, 4930722]
- Chinese University of Hong Kong [4930722]
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With the emergence of multi- and extensive-drug (MDR/XDR) resistant Mycobacterium tuberculosis (M. tb), tuberculosis (TB) persists as one of the world's leading causes of death. Recently, isothermal DNA amplification methods received much attention due to their ease of translation onto portable point-of-care (POC) devices for TB diagnosis. In this study, we aimed to devise a simple yet robust detection method for M. tb. Amongst the numerous up-and-coming isothermal techniques, Recombinase Polymerase Amplification (RPA) was chosen for a real-time detection of TB with or without MDR. In our platform, real-time RPA (RT-RPA) was integrated on a lab on-a-disc (LOAD) with on-board power to maintain temperature for DNA amplification. Sputa collected from healthy volunteers were spiked with respective target M. tb samples for testing. A limit of detection of 102 colony-forming unit per millilitre in 15 min was achieved, making early detection and differentiation of M. tb strains highly feasible in extreme POC settings. Our RT-RPA LOAD platform has also been successfully applied in the differentiation of MDR-TB from H37Ra, an attenuated TB strain. In summary, a quantitative RT-RPA on LOAD assay with a high level of sensitivity was developed as a foundation for further developments in medical bedside and POC diagnostics.
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