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Mol Cell Probes. 2015 Apr;29(2):92-8. doi: 10.1016/j.mcp.2014.12.001. Epub 2014 Dec 24.

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

Author information

1
LGC, Queens Road, Teddington, TW11 0LY, UK.
2
University of Southampton School of Chemistry, Highfield, Southampton SO17 1BJ, UK.
3
University of Southampton Faculty of Medicine, Clinical and Experimental Sciences, Southampton General Hospital, Southampton SO16 6YD, UK.
4
OptiGene Ltd., Unit 5, Blatchford Road, Horsham, RH13 5QR, UK.
5
Health Protection Agency, Southampton General Hospital, Southampton SO16 6YD, UK.
6
LGC, Queens Road, Teddington, TW11 0LY, UK. Electronic address: paul.debenham@lgcgroup.com.

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.

KEYWORDS:

CPA; HyBeacons; Isothermal amplification; LAMP; Probes; SMAP

PMID:
25542839
DOI:
10.1016/j.mcp.2014.12.001
[Indexed for MEDLINE]

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