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

Overview

Motivation

Background

U-dHRM Platform

Current Work

Summary

Acknowledgements

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.

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!

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

Background – Molecular Diagnostics

DNA Microarrays

Quantitative Real-time PCR

Rapid, no need for culture

Lacks broad-based detection

Hybridization inaccuracies

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

Background – High Resolution Melt

Rapid, inexpensive post-PCR

sequencing alternative

Non-specific intercalating dye

Solely dependent on sequence

Constrained to homogeneous samples

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

Universal Digital HRM (U-dHRM)

16S PCR broad-based, rapid

Digital PCR / Microfluidics sensitive, polymicrobial

High Resolution Melt cost-effective

U-dHRM Platform – Workflow

DNA Isolation dPCR Chip Sequestration

16S Gene Amplification

Heat RampFluorescence AnalysisMelt Curve Analysis

U-dHRM Platform – Heating System

Heat Sink

Copper Block

Peltier Chip

Microscope

Stage Adapter

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

Current Work – Brief

Transition to digital droplet PCR

Scalable dynamic range

High-throughput

Portability

Image entire chip

Remove microscope

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

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