extreme molecular diagnosticstesting times (from the physician/patient viewpoint) reference labs...
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Extreme Molecular Diagnostics
Carl Wittwer, Department of Pathology, University of Utah
ARUP, Oct 22, 2019, Salt Lake City, UT
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How to Innovate:
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Outline(our focus is speed)
• Current state of the art– Sample preparation, amplification, analysis
• Making amplification faster– Rapid-cycle PCR
– Extreme PCR
• Making analysis faster– High speed melting
• Making sample preparation faster– Genomic DNA from whole blood
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Rapid Targeted Molecular Assays(Flu A/B, RSV, Strep A)
• Real-time PCR
– 15-30 minutes
– Multiple manufacturers
• Recombinase polymerase assay
– Isothermal
– Positive results in 2-5 min
– Negative results in 6-13 min
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Multiplex Syndromic Tests
(FDA-approved)
Panel Pathogens
(#)
Resistance
Targets (#)
Time to Result
(min)
Respiratory 21 45
Blood Culture ID 24 3 60
Gastrointestinal 22 60
Meningitis 14 60
Pneumonia 26 7 60
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Microbial Cell-free DNA SequencingNat Microbiol 2019, 4, 663-674
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Clinical Genome Sequencing (Pediatric ICU)Sci Transl Med (2019, 11, 6177)
• 20 hour whole genome sequencing– 1.5 hours of library preparation
– 15.5 hours massively parallel sequencing
– 1 hour of alignment and variant calling
• Automated phenotyping and interpretation– Phenome extraction from electronic health record
– Match to phenomes of all genetic diseases
– Correlate to pathogenic variants
• Guinness World Record for Fastest Genetic Diagnosis
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Making PCR Faster
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1985-1988: DNA replication in a test tube
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Trouble with Terminology
PCR Era 30 Cycles Year
Legacy 2-4 hours 1989
Rapid Cycle 10-30 min 1991
Fast 30 min-1 hour 2000s
Ultrafast 2-10 min 2010s
Extreme <15-60 sec 2015
• “Rapid”, “Fast” are relative
• “almost instantaneous”
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Sample Temperatures in PCR
Conventional
Cycling
Time (min)
Sam
ple
Tem
pe
ratu
re (
°C)
Time (min)
Rapid
CyclingSam
ple
Tem
pera
ture
(°C
)
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Rapid Cycling is More Specific
Amplification of a 536 bp -globin fragment
from human genomic DNATime (min)
Sam
ple
Tem
pera
ture
(°C
)
Gel
AnalysisTime for 30
Cycles (hr)Temperature Profiles
536 bp
Anal Biochem 1990;186:328-31, Biotechniques. 1991;10:76-83
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Rapid Cycling Instrument
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Other Containers for Rapid PCR
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Ethidium Bromide / Transilluminator
Monitoring PCR with
Fluorescence
Flow Cytometry
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Monitoring Fluorescence during
Amplification
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RapidCycler + Fluorimeter
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Real-Time Prototype
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How long does it take to….
• Denature• Fast! (<1 sec)
• Anneal• Depends on the primer concentration
• Extend• Complex
• Depends on the speed and concentration of polymerase
• 5 ms for each nucleotide addition
• 50 ms for binding events
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Extreme PCR
10X
Primers10X
Polymerase
10X Speed
10X
Products
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HOT
WATER
COLD
WATER
Sample
Holder
Capillaries
Stepper
Motor
Optics
Fiber
Optical
Stage
Real Time PCR Extreme Alpha Prototype
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Water Bath Prototypes for
Extreme Real-Time PCR
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12 min PCR30 sec PCR
Extreme PCR compared to Rapid Cycle PCR(45 bp human genomic target KCNE1)
50 bp
100 bp
Primers
NTC NTC
Extreme
PCR
(28 sec)
Rapid Cycle
PCR
(12 min)
[Polymerase] (µM) 1 0.064
[Primers] (µM) 10 0.5
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Polymerase and Primer Optimization NQO1 (102 bp)58 sec PCR (30 cycles, 1.93 sec/cycle)
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y = -3.538x + 39.231R² = 0.9922
20
25
30
35
40
0 1 2 3 4Qu
an
tifi
ca
tio
n c
yc
le
(Cq
)
Log10(initial template copies)
y = -3.64x + 38.236R² = 0.9909
20
25
30
35
40
0 1 2 3 4
Qu
an
tifi
ca
tio
n c
yc
le
(Cq
)
Log10(initial template copies)
Extreme PCR Efficiency and Sensitivity
91.7% (45 bp, 28 sec PCR) 95.8% (102 bp, 58 sec PCR)
0
20
40
60
0 10 20 30 40 50
Flu
ore
sc
en
ce
Cycle number
15000
1500
150
15
1.5
NTC
0
20
40
60
80
0 10 20 30 40 50
Flu
ore
sc
en
ce
Cycle Number
15000
1500
150
15
1.5
NTC
Copies15,000
1,500
150
15
1.5
NTC
Copies15,000
1,500
150
15
1.5
NTC
Clin Chem. 2015 Jan;61(1):145-53
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50 bp
25 bp
75 bp
11.2 s 14.7 s 18.2 s 21.7 s 21.7 s
NTC
PCR Time
14.7 second PCR 60 bp AKAP10 (35 cycles, 0.42 sec/cycle)
Clin Chem 2015;61:145-53
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Lessons from making PCR faster
• Slow PCR is an accident of history• Limited instrumentation
• Slow cycling requires low reagent concentrations
• High reagent costs
• Science is fair• Never been “scooped”
• Close calls
• The market values:• Numbers over quality
• Convenience over speed• Capillaries
• Water baths
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Extreme PCR on a microfluidic
system
Clin Chem. 2019 Feb;65(2):263-271.
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Making Analysis Faster
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Nucleic Acid Analysis
• Electrophoresis• Separation matrix
• Reveals size differences
• Mass Spectroscopy
• HPLC
• Sequencing by synthesis
• DNA melting• Solution technique
• No additions or separations
• Reveals melting profile differences
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Modern melting analysis is performed after PCR
• Advances– Sensitivity
• Fluorescence instead of Absorbance
– Cost• Dyes vs Probes
– Speed…..
Cycle Number
Flu
ore
scen
ce
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Tim
e (
min
)
Temperature (°C)
Dynamic Dot Blot for Genotyping(labeled probes)
Anchor Probe Mutation Probe
Match
Mismatch
Flu
ore
sc
en
ce
Temperature (°C)
Temperature (°C)
-dF
/dT
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Dual Hybridization Probes
Am J Pathol. 1998;153:1055-61
Single Hybridization Probe
Anal Biochem. 2001;290:89-97
Unlabeled Probe
Clin Chem. 2004;50:1328-35
Genotyping by Melting
***Two probes identify many alleles******One probe identifies many alleles***
Snapback Primer
Clin Chem. 2008;54:1648-56
Variant
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Genotyping by Small Amplicon Melting(dyes)
DTm
Clin Chem 50: 1156 – 64, 2004
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High Resolution Melting(2 min)
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High Resolution Melting(Rates and Times)
Instrument Recommended
Setting
Measured Ramp Rate
(°C/s)
Melting
Time (min)
A Step 0.04°C
Hold 1 s
0.01 40
B Ramp 0.1°C
Hold 2 s
0.01 40
C Step 0.2°C
Hold 10 s
0.01 50
D 0.3% Ramp 0.005 95
Clin Chem. 2014 Jun;60(6):864-72
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Amplicon Melting as PCR Quality Control
• Bad PCR?• Expect a single transition
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Melting Curve Prediction(uMelt: dna.utah.edu)
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Faster SNV Melting Rates Improve
Genotype Resolution
Anal Biochem 2017;539:90-95
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Clin Chem 2017;63:1624-32
Microfluidic High Speed Melting
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Rapid Cycle vs Extreme PCR
1996 – Rapid Cycling 2018 - Microfluidics
(28 seconds/cycle) (1.05 seconds/cycle)
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Making Sample Preparation
Faster
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Nucleic Acid Preparation
• Depends on the matrix
– Blood, chicken, anthrax, woolly mammoth
• Depends on the target
– RNA, DNA
• Some sample types require no purification
– Swabs (respiratory/pharyngeal)
– Thermal cycling only
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Genomic DNA from Blood
• DNA release from histones
– Chaotropes
– Enzymes
• 30 min – 2 hours
– Most manual kits
– Most automated systems
• 15 min
– Single tube digestion
– Temperature control
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DNA Extraction from Blood with NaOH(lye for lysis)
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Quantitative DNA release from
blood with NaOH
• Limiting dilution analysis
• WBC
• 0.2 cells/well = 0.8 strands/well
• 58/96 wells positive
• 0.93 strands/well
• 115% recovery
• 84 – 146% recovery (95% confidence)
Fast Complete
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Inhibition of fluorescence
with constant efficiency
Eventual inhibition of efficiency
Real-time monitoring of NaOH-treated whole blood
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Melting analysis from NaOH-lysed whole blood
(rs1024116)
SMN1 Reference
Small Amplicon Genotyping
1
2
3
A/G
A/A G/G
Copy Number(SMA – spinal muscular atrophy)
Clin Chem 50:1156-64;2004 Clin Chem 61:724-33;2015
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Clinical lab tests from a single drop of bloodBlood drop = 46 +/- 5 µL
• 5,000 WBC/µL
• 20,000 PCR templates/µL
• 25-fold dilution in NaOH
• 800 templates/µL
• 10-fold dilution into PCR
• 80 templates/µL
• Five µL PCR
• 400 templates
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Can we go from a finger prick to real-time detection in < 1 min?
• Human blood
• Single copy gene
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Testing Times(from the physician/patient viewpoint)
Reference Labs Point-of-Care
Pre-analytical >12 hours Fast!
Analytical (varies) (varies)
Post-analytical ~8 hours Fast!
• Point of care eliminates most pre- and post analytical steps
• Rapid testing has limited value for reference labs
• Rapid testing is critical for point-of-care value
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Summary
• Extreme PCR– Increase speed 200X
– Efficient, sensitive, and specific
• High Speed Melting
– Increase 100-1000X over conventional melting
• Extreme sample preparation
– In seconds
• Faster is better (PCR and melting)
• Chemicals and enzymes are fast, people and their machines are slow
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Thanks!BioFire / bioMerieux
Kirk Ririe
Randy Rasmussen
NIH
ARUP
Roche Applied Science
Canon
State of Utah
University of Utah
Mark Herrmann
Jared Farrar
Luming Zhou
Rob Pryor
Adam Millington
Felix Ye
Website: https://www.dna.utah.edu