David French, LGC - MMS Conferencing

HyBeacon ProbesGenetic applications at thepoint-of-careDr David French

Traditional vs point-of-carediagnostics• Sample taken at Doctor’ssurgery or hospital• Often invasive blood andswab samples• Laboratory analysis• Results back to Doctor• Results to patient• Diagnosis sometimes 1week after initial consultation• Inappropriate drugs may beemployed if treated beforediagnosis• Sample provided at Doctor’ssurgery, hospital orpharmacy• Non-invasive saliva andurine samples• On-site analysis within 20-30minutes• Diagnosis and appropriatetreatment• Correct drug, correct dosageand avoidance of adversereactions

Rapid sample analysis• Sample preparation• PCR amplification• PCR product detection andidentification• Results analysisEliminateSpeed up and detect targetDNA simultaneouslyHomogeneous PCRAutomate

• ClinicalApplications– Genetic diagnosis– Infectious disease detection• Pharmaceutical & Pharmacogenetics– Polymorphisms associated with drug metabolism• Food– Species identification– GM and pathogen detection– Selective breeding (e.g. Scrapie)• Forensic– Scene-of-crime, point-of-arrest• Defence– Bio-threat detection

HyBeacon probes• Fluorescent probe technology• Benefits over existing commercialsystems5´5´3´• Single probe to analysehomozygous and heterozygoussamples• The quenching properties of DNAreduces single-stranded probeemission3´5´3´• Fluorescence increases upon targethybridisation

Differential probe hybridisation• HyBeacon probes are significantly more stable when hybridisedto fully complementary sequences• Discrimination of closely related sequences on the basis ofmelting temperature (Tm)• Tm = 50% of probe hybridised to target• Clearly differentiates DNA sequences varying by as singlenucleotide

HyBeacon fluorescence &sequence analysis5´5´3´3´

Direct sample analysis• Purification of samples not required• Suitable for point-of-care systems• Achieved target amplification anddetection directly from– Saliva– Urine– Swabs– Blood

Ultra-rapid detection and discrimination of NAT2polymorphic targets from saliva*5C/*5C*4/*5C *4/*4M*4/*4 *4/*5C *5C/*5C M121bp98bp58bp23bpTm = 42.3°CTm = 42.6°C + 52.5°CTm = 52.4°C

Factor V Leiden• SNP in factor V gene stronglyassociated with increasedrisk for:– deep vein thrombosis(DVT)– pulmonary embolism• 5-10 and 50-100 foldincreased risk inheterozygous andhomozygous individualsrespectively

Analysis of factor V LeidenN18 G2 N18 G3 NTC

Pathogen detection• Chlamydia trachomatis• Infections can be asymptomaticand may cause sterility ifuntreated• High costs to health service• Patient may not return for resultsand treatment if diagnosis is slow• Point-of-care facilities mayimprove treatment rate• Efficient HyBeacon assaydeveloped capable of detectingpathogen in urine and swabsamples

Automated sample analysis• Positive amplification control required to distinguish negativesamples from PCR failsChlamydia peakgenerated in negativecontrolNo peaksgeneratedBoth peaksgeneratedOnly control peakgenerated• Single nucleotide substitution introduced into mimic construct• Target and mimic amplified and detected with same primersand probeTEST FAILINFECTEDUNINFECTED

Thermal Cycler• Genedrive• Single use plasticconsumables• Developing simple to usetouch screen instrument• 50 cycles of PCR and meltanalysis within 30 minutes• Suitable for point-of-caretests295mm330mm

Patient sampleDiagnosis &treatmentCartridgescontainingfreeze-driedreagents20 minutes processing

High Throughput screening• Melt analysis also possible in 96 and 384well plate formats• Sheep prion protein (PrP) gene mutations(Scrapie)

Summary• Novel fluorescent probe technology• Rapid sequence analysis– Pharmacogenetics– Disease predisposition– Healthcare– Pathogen detection• Non-invasive tests with point-of care application• Also suited to high throughput screening

Acknowledgements• LGC– David McDowell– Gavin Nixon– Rajinder Flora– Paul Debenham• Osmetech– John Clarkson• University of Southampton– Professor Tom Brown