Recommended
More Related Content
What's hot
What's hot (20)
Similar to Benjamin Yang -- SRC Presentation
Similar to Benjamin Yang -- SRC Presentation (20)
Benjamin Yang -- SRC Presentation
- 1. High-Throughput Universal Digital High-Resolution Melt (U-dHRM)Platform for Bacterial Identification BENJAMIN YANG UC SCHOLARS SUMMER RESEARCH CONFERENCE 2016 UNIVERSITY OF CALIFORNIA, SAN DIEGO
- 2. Overview Motivation Background U-dHRM Platform Current Work Summary Acknowledgements
- 3. Motivation – Sepsis >750,000 cases in the US each year 26% - 50% mortality rate One of top 10 causes of infant mortality Tricky to diagnose Non-specific symptoms Unhelpful diagnostic protocols Overwhelming and life-threatening inflammatory response to a bloodstream infection.
- 4. Motivation – Blood Culture Current gold standard Can take several days or more for a result Difficult to determine bacterial strains Broad-spectrum antibiotics in the interim Negative results don’t necessarily affect diagnosis!
- 5. Motivation – Diagnostic Criteria Broad-based – detect all bacteria in sample Sensitive – small sample volumes and 1 – 2000 CFU/mL Rapid – targeted antibiotic treatment based on specific strains Polymicrobial – precise composition of heterogeneous samples
- 6. Background – Molecular Diagnostics DNA Microarrays Quantitative Real-time PCR Rapid, no need for culture Lacks broad-based detection Hybridization inaccuracies
- 7. Background – 16S PCR 16S rRNA gene Present in all bacteria broad-based Strain-specific hypervariable regions Prone to false positives and negatives from small volumes (1-5 uL) Still need to sequence amplicon
- 8. Background – High Resolution Melt Rapid, inexpensive post-PCR sequencing alternative Non-specific intercalating dye Solely dependent on sequence Constrained to homogeneous samples
- 9. Background – dPCR Scalable dynamic range and cost-effective DNA occupancy follows a Poisson distribution 10 - 100 wells to DNA molecules 96 wells up to 9 molecules 20,000 wells up to 2,000 molecules Neonate blood contains 1-2,000 CFU/mL Partitions sample into individual reaction wells polymicrobial & sensitive
- 10. Universal Digital HRM (U-dHRM) 16S PCR broad-based, rapid Digital PCR / Microfluidics sensitive, polymicrobial High Resolution Melt cost-effective
- 11. U-dHRM Platform – Workflow DNA Isolation dPCR Chip Sequestration 16S Gene Amplification Heat RampFluorescence Analysis Melt Curve Analysis
- 12. U-dHRM Platform – Heating System Heat Sink Copper Block Peltier Chip Microscope Stage Adapter
- 13. U-dHRM – One-versus-one Support Vector Machine (OVO SVM) Supervised – learns from labeled training data (support vectors) Binary – determines between 2 classifications L. monocytogenes S. pneumoniae Unknown Curves Training Data Classified Curves
- 14. Current Work – Brief Transition to digital droplet PCR Scalable dynamic range High-throughput Portability Image entire chip Remove microscope
- 15. Summary U-dHRM Platform Broad-based – universal 16S amplification Polymicrobial – highly accurate profiling of heterogeneous samples Sensitive – dPCR chip Rapid – DNA amplification strategies and HRM Current Work Provides high-throughput format Improves portability for clinical applications
- 16. Acknowledgments Dr. Colleen Chute-Ricker Dr. Julietta Jupe Daniel Ortiz Tyler Goshia Anthony Han Hannah Mack Nick Teuthorn Many thanks to all the members of the Fraley Lab Many thanks to our collaborators Dr. Shelley Lawrence – Assistant Professor, Pediatrics, UCSD Medicine Dr. Hannah Carter – Assistant Professor, Medical Genetics, UCSD Medicine Brian Tsui – Carter Lab Mridu Sinha – Coleman Lab Many thanks to our sponsors