Abstract
It is challenging to employ nucleic acid-based diagnostics for the in situ detection of Clostridium difficile from complex fecal samples because essential sample preparation and amplification procedures require various experimental resources. In this study, a simple and effective on-site nucleic acid-based detection system was used to detect C. difficile in stool samples. Two columns containing different microbeads, namely, glass and functionalized graphene oxide-coated microbeads, were designed to remove relatively large impurities by filtration and concentrate bacteria, including C. difficile, from stool samples by adsorption. The bacterial nucleic acids were effectively extracted using a small bead beater. The effectiveness of enzyme inhibitors remaining in the sample was efficiently reduced by the direct buffer developed in this study. This sample preparation kit consisting of two simple columns showed better performance in real-time polymerase chain reaction (PCR) and equivalent performance in loop-mediated isothermal amplification (LAMP) than other sample preparation kits, despite 90% simplification of the process. The amplification-ready samples were introduced into two microtubes containing LAMP pre-mixtures (one each for E. coli as an external positive control and C. difficile) by a simple sample loader, which was operated using a syringe. LAMP, which indicates amplification based on color change, was performed at 65 °C in a small water bath. The limit of detection (L.O.D) and analytical sensitivity/specificity of our simple and effective kit were compared with those of a commercial kit. C. difficile in stool samples could be detected within 1 h with 103 cfu/10 mg using LAMP combined simple on-site detection kit.
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Funding
This work was supported by the Technology Innovation Program (or Industrial Strategic Technology Development Program) (20008702, development of automated non-invasive sample preparation system for digital healthcare) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea), and supported by the Korea Medical Device Development Fund grant funded by the Korean Government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (Project Number: NTIS No. 1711134988, KMDF_PR_20200901_0020-2021-02) and by the government-wide R&D Fund project for infectious disease research (GFID), Republic of Korea (Grant Number: HG18C0012).
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All experiments on stool samples were approved by the ethics committee of Cheju Halla General Hospital (CHH 2020-D04-1). Informed consents were obtained from human participants of this study.
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Baek, C., Li, Y.G., Yoo, H.J. et al. Simple and portable on-site system for nucleic acid-based detection of Clostridium difficile in stool samples using two columns containing microbeads and loop-mediated isothermal amplification. Anal Bioanal Chem 414, 613–621 (2022). https://doi.org/10.1007/s00216-021-03557-4
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DOI: https://doi.org/10.1007/s00216-021-03557-4