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Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes – a homogenous system for sequence-specific detection

Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes – a homogenous system for sequence-specific detection
Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes – a homogenous system for sequence-specific detection
Isothermal amplification is a rapid, simple alternative to PCR, with amplification commonly detected using fluorescently labelled oligonucleotide probes, intercalating dyes or increased turbidity as a result of magnesium pyrophosphate generation. SNP identification is possible but requires either allele-specific primers or multiple dye-labelled probes, but further downstream processing is often required for allelic identification. Here we demonstrate that modification of common isothermal amplification methods by the addition of HyBeacon probes permits homogeneous sequence detection and discrimination by melting or annealing curve analysis. Furthermore, we demonstrate that isothermal amplification and sequence discrimination is possible directly from a crude sample such as an expressed buccal swab.
HyBeacons, isothermal amplification, probes, LAMP, SMAP, CPA
0890-8508
92-98
Howard, Rebecca L.
a23d7650-7143-485a-bba7-d417cb3cdc0c
French, David J.
019c0355-a863-48a4-a21b-f534d2da96b4
Richardson, James A.
51db9f73-a136-48af-8722-21d46906cf40
O'Neill, Colette E.
3de0c221-6578-4a1a-96bd-2a3fba2b6193
Andreou, Michael P.
4f47d5f6-8145-4ed8-b13c-00cbc6e5cd38
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Clark, Duncan
262201c6-5eaa-4f0d-b778-c0a40b227a7b
Clarke, Ian N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Holloway, John W.
4bbd77e6-c095-445d-a36b-a50a72f6fe1a
Marsh, Peter
2f77a131-1871-45b8-a5d8-a5ea9beb430a
Debenham, Paul G.
ae162b2d-e340-48a9-ac98-849d3466438f
Howard, Rebecca L.
a23d7650-7143-485a-bba7-d417cb3cdc0c
French, David J.
019c0355-a863-48a4-a21b-f534d2da96b4
Richardson, James A.
51db9f73-a136-48af-8722-21d46906cf40
O'Neill, Colette E.
3de0c221-6578-4a1a-96bd-2a3fba2b6193
Andreou, Michael P.
4f47d5f6-8145-4ed8-b13c-00cbc6e5cd38
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Clark, Duncan
262201c6-5eaa-4f0d-b778-c0a40b227a7b
Clarke, Ian N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
Holloway, John W.
4bbd77e6-c095-445d-a36b-a50a72f6fe1a
Marsh, Peter
2f77a131-1871-45b8-a5d8-a5ea9beb430a
Debenham, Paul G.
ae162b2d-e340-48a9-ac98-849d3466438f

Howard, Rebecca L., French, David J., Richardson, James A., O'Neill, Colette E., Andreou, Michael P., Brown, Tom, Clark, Duncan, Clarke, Ian N., Holloway, John W., Marsh, Peter and Debenham, Paul G. (2015) Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes – a homogenous system for sequence-specific detection. Molecular and Cellular Probes, 29 (2), 92-98. (doi:10.1016/j.mcp.2014.12.001). (PMID:25542839)

Record type: Article

Abstract

Isothermal amplification is a rapid, simple alternative to PCR, with amplification commonly detected using fluorescently labelled oligonucleotide probes, intercalating dyes or increased turbidity as a result of magnesium pyrophosphate generation. SNP identification is possible but requires either allele-specific primers or multiple dye-labelled probes, but further downstream processing is often required for allelic identification. Here we demonstrate that modification of common isothermal amplification methods by the addition of HyBeacon probes permits homogeneous sequence detection and discrimination by melting or annealing curve analysis. Furthermore, we demonstrate that isothermal amplification and sequence discrimination is possible directly from a crude sample such as an expressed buccal swab.

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More information

e-pub ahead of print date: 24 December 2014
Published date: April 2015
Keywords: HyBeacons, isothermal amplification, probes, LAMP, SMAP, CPA
Organisations: Human Development & Health, Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 373094
URI: https://eprints.soton.ac.uk/id/eprint/373094
ISSN: 0890-8508
PURE UUID: d9a4ef1f-37dc-4e01-8dc5-c16e4688515d
ORCID for Ian N. Clarke: ORCID iD orcid.org/0000-0002-4938-1620
ORCID for John W. Holloway: ORCID iD orcid.org/0000-0001-9998-0464

Catalogue record

Date deposited: 06 Jan 2015 16:59
Last modified: 06 Jun 2018 13:19

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