Development of an on-disc isothermal in vitro amplification and detection of bacterial RNA

Des Brennan; Helena Coughlan; Eoin Clancy; Nikolay Dimov; Thomas Barry; David Kinahan; Jens Ducrée; Terry J. Smith; Paul Galvin

doi:10.1016/j.snb.2016.08.018

Development of an on-disc isothermal in vitro amplification and detection of bacterial RNA

Des Brennan, Helena Coughlan, Eoin Clancy, Nikolay Dimov, Thomas Barry, David Kinahan, Jens Ducrée, Terry J. Smith, Paul Galvin

School of Physics, Engineering & Computer Science

Research output: Contribution to journal › Article › peer-review

15 Citations (Scopus)

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Abstract

We present a centrifugal microfluidic “Lab-on-a-Disc” (LoaD) system capable of implementing nucleic acid in vitro amplification using non-contact heating and fluorescence detection. The system functionality is verified by implementing a Nucleic Acid Sequence Based Amplification (NASBA) reaction, targeting the tmRNA transcript of Haemophilus influenzae. The NASBA assay incorporates fluorescent molecular beacon probes reporting target tmRNA amplification for endpoint detection. The system implements non-contact IR heating to heat the NASBA reaction to the required target temperatures during denaturation and amplification steps. The LoaD control system facilitates spin speed and chamber positioning for heating and fluorescence detection. The LoaD alignment system uses magnetic fields to locate and lock the chamber in the required position (heating or detection). The NASBA assay was implemented on the system using Haemophilus influenzae tmRNA over the range 10²–10⁴ cell equivalent (CE) units. For comparison, identical qNASBA assays were implemented on a Roche LightCycler 2.0 over this concentration range.

Original language	English
Pages (from-to)	235-242
Number of pages	8
Journal	Sensors and Actuators, B: Chemical
Volume	239
Early online date	3 Aug 2016
DOIs	https://doi.org/10.1016/j.snb.2016.08.018
Publication status	Published - 1 Feb 2017

Keywords

Fluorescence detection
IR heating
Isothermal amplification
Lab-on-a-Disc(LoaD)
tmRNA

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10.1016/j.snb.2016.08.018

Accepted ManuscriptAccepted author manuscript, 935 KBLicence: CC BY-NC-ND

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title = "Development of an on-disc isothermal in vitro amplification and detection of bacterial RNA",

abstract = "We present a centrifugal microfluidic “Lab-on-a-Disc” (LoaD) system capable of implementing nucleic acid in vitro amplification using non-contact heating and fluorescence detection. The system functionality is verified by implementing a Nucleic Acid Sequence Based Amplification (NASBA) reaction, targeting the tmRNA transcript of Haemophilus influenzae. The NASBA assay incorporates fluorescent molecular beacon probes reporting target tmRNA amplification for endpoint detection. The system implements non-contact IR heating to heat the NASBA reaction to the required target temperatures during denaturation and amplification steps. The LoaD control system facilitates spin speed and chamber positioning for heating and fluorescence detection. The LoaD alignment system uses magnetic fields to locate and lock the chamber in the required position (heating or detection). The NASBA assay was implemented on the system using Haemophilus influenzae tmRNA over the range 102–104 cell equivalent (CE) units. For comparison, identical qNASBA assays were implemented on a Roche LightCycler 2.0 over this concentration range.",

keywords = "Fluorescence detection, IR heating, Isothermal amplification, Lab-on-a-Disc(LoaD), tmRNA",

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AU - Brennan, Des

AU - Coughlan, Helena

AU - Clancy, Eoin

AU - Dimov, Nikolay

AU - Barry, Thomas

AU - Kinahan, David

AU - Ducrée, Jens

AU - Smith, Terry J.

AU - Galvin, Paul

N1 - This document is the Accepted Manuscript version, made available under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License CC BY NC-ND 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/). The final, published version is available online at doi: https://doi.org/10.1016/j.snb.2016.08.018. Published by Elsevier B. V.

PY - 2017/2/1

Y1 - 2017/2/1

N2 - We present a centrifugal microfluidic “Lab-on-a-Disc” (LoaD) system capable of implementing nucleic acid in vitro amplification using non-contact heating and fluorescence detection. The system functionality is verified by implementing a Nucleic Acid Sequence Based Amplification (NASBA) reaction, targeting the tmRNA transcript of Haemophilus influenzae. The NASBA assay incorporates fluorescent molecular beacon probes reporting target tmRNA amplification for endpoint detection. The system implements non-contact IR heating to heat the NASBA reaction to the required target temperatures during denaturation and amplification steps. The LoaD control system facilitates spin speed and chamber positioning for heating and fluorescence detection. The LoaD alignment system uses magnetic fields to locate and lock the chamber in the required position (heating or detection). The NASBA assay was implemented on the system using Haemophilus influenzae tmRNA over the range 102–104 cell equivalent (CE) units. For comparison, identical qNASBA assays were implemented on a Roche LightCycler 2.0 over this concentration range.

AB - We present a centrifugal microfluidic “Lab-on-a-Disc” (LoaD) system capable of implementing nucleic acid in vitro amplification using non-contact heating and fluorescence detection. The system functionality is verified by implementing a Nucleic Acid Sequence Based Amplification (NASBA) reaction, targeting the tmRNA transcript of Haemophilus influenzae. The NASBA assay incorporates fluorescent molecular beacon probes reporting target tmRNA amplification for endpoint detection. The system implements non-contact IR heating to heat the NASBA reaction to the required target temperatures during denaturation and amplification steps. The LoaD control system facilitates spin speed and chamber positioning for heating and fluorescence detection. The LoaD alignment system uses magnetic fields to locate and lock the chamber in the required position (heating or detection). The NASBA assay was implemented on the system using Haemophilus influenzae tmRNA over the range 102–104 cell equivalent (CE) units. For comparison, identical qNASBA assays were implemented on a Roche LightCycler 2.0 over this concentration range.

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KW - IR heating

KW - Isothermal amplification

KW - Lab-on-a-Disc(LoaD)

KW - tmRNA

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JO - Sensors and Actuators, B: Chemical

JF - Sensors and Actuators, B: Chemical

ER -

Development of an on-disc isothermal in vitro amplification and detection of bacterial RNA

Abstract

Keywords

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