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Real-time microfluidic recombinase polymerase amplification for the toxin B gene of clostridium difficile on a SlipChip platform

Real-time microfluidic recombinase polymerase amplification for the toxin B gene of clostridium difficile on a SlipChip platform
Real-time microfluidic recombinase polymerase amplification for the toxin B gene of clostridium difficile on a SlipChip platform
Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 ?L. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents.
0003-2654
Tsaloglou, M.-N.
99ab30ba-15da-4d25-86ba-608d127f8369
Watson, R.J.
337ffd04-4e35-4bb1-a07e-d6a898ccb646
Rushworth, C.M.
633f1246-5650-415e-b3a4-7e6cfa5e4159
Zhao, Yi
0d8feab3-da2f-4efe-a099-2fa7c7c9f31e
Niu, Xize
f3d964fb-23b4-45db-92fe-02426e4e76fa
Sutton, J.M.
481fee15-5bb6-4a25-a7ee-289e821d662e
Morgan, H.
de00d59f-a5a2-48c4-a99a-1d5dd7854174
Tsaloglou, M.-N.
99ab30ba-15da-4d25-86ba-608d127f8369
Watson, R.J.
337ffd04-4e35-4bb1-a07e-d6a898ccb646
Rushworth, C.M.
633f1246-5650-415e-b3a4-7e6cfa5e4159
Zhao, Yi
0d8feab3-da2f-4efe-a099-2fa7c7c9f31e
Niu, Xize
f3d964fb-23b4-45db-92fe-02426e4e76fa
Sutton, J.M.
481fee15-5bb6-4a25-a7ee-289e821d662e
Morgan, H.
de00d59f-a5a2-48c4-a99a-1d5dd7854174

Tsaloglou, M.-N., Watson, R.J., Rushworth, C.M., Zhao, Yi, Niu, Xize, Sutton, J.M. and Morgan, H. (2014) Real-time microfluidic recombinase polymerase amplification for the toxin B gene of clostridium difficile on a SlipChip platform. Analyst. (doi:10.1039/C4AN01683A).

Record type: Article

Abstract

Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 ?L. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents.

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Published date: 23 October 2014
Organisations: Electronics & Computer Science

Identifiers

Local EPrints ID: 368171
URI: https://eprints.soton.ac.uk/id/eprint/368171
ISSN: 0003-2654
PURE UUID: efe37210-d31e-46bf-bb76-8aa89f187d83
ORCID for H. Morgan: ORCID iD orcid.org/0000-0003-4850-5676

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Date deposited: 19 Aug 2014 12:56
Last modified: 10 Dec 2019 01:46

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Contributors

Author: M.-N. Tsaloglou
Author: R.J. Watson
Author: C.M. Rushworth
Author: Yi Zhao
Author: Xize Niu
Author: J.M. Sutton
Author: H. Morgan ORCID iD

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