title: Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes--a homogenous system for sequence-specific detection.
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.
authors.affiliation: LGC, Queens Road, Teddington, TW11 0LY, UK.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Howard
authors.affiliation: LGC, Queens Road, Teddington, TW11 0LY, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: French
authors.affiliation: University of Southampton School of Chemistry, Highfield, Southampton SO17 1BJ, UK.
authors.forename: James A
authors.initials: JA
authors.lastname: Richardson
authors.affiliation: University of Southampton Faculty of Medicine, Clinical and Experimental Sciences, Southampton General Hospital, Southampton SO16 6YD, UK.
authors.forename: Colette E
authors.initials: CE
authors.lastname: O'Neill
authors.affiliation: OptiGene Ltd., Unit 5, Blatchford Road, Horsham, RH13 5QR, UK.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Andreou
authors.affiliation: University of Southampton School of Chemistry, Highfield, Southampton SO17 1BJ, UK.
authors.forename: Tom
authors.initials: T
authors.lastname: Brown
authors.affiliation: OptiGene Ltd., Unit 5, Blatchford Road, Horsham, RH13 5QR, UK.
authors.forename: Duncan
authors.initials: D
authors.lastname: Clark
authors.affiliation: University of Southampton Faculty of Medicine, Clinical and Experimental Sciences, Southampton General Hospital, Southampton SO16 6YD, UK.
authors.forename: Ian N
authors.initials: IN
authors.lastname: Clarke
authors.affiliation: University of Southampton Faculty of Medicine, Clinical and Experimental Sciences, Southampton General Hospital, Southampton SO16 6YD, UK.
authors.forename: John W
authors.initials: JW
authors.lastname: Holloway
authors.affiliation: Health Protection Agency, Southampton General Hospital, Southampton SO16 6YD, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Marsh
authors.affiliation: LGC, Queens Road, Teddington, TW11 0LY, UK. Electronic address: paul.debenham@lgcgroup.com.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Debenham
mesh_terms: D002692:Chlamydia trachomatis / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D015335:Molecular Probes / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods; D064417:Vitamin K Epoxide Reductases / Q000235:genetics
chemical_list: D015335:Molecular Probes; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases
keywords: CPA; HyBeacons; Isothermal amplification; LAMP; Probes; SMAP