Ebola IMDx
16
Allison Reichl TOWARDS AN INTEGRATED MOLECULAR DIAGNOSTIC SYSTEM FOR EBOLA VIRUS MOCK
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Transcript of Ebola IMDx
Allison Reichl
TOWARDS AN INTEGRATED MOLECULAR DIAGNOSTIC
SYSTEM FOR EBOLA VIRUS
MOCK
IntroductionDevice design
Thermal Lysis of Ebola viral particles Off-chip thermal lysis
In water Optimization in plasma
On-chip photothermal lysisRT-PCR of Ebola viral particles
Comparison of Benchtop and Optical Cavity RT-PCR SpinColumn Kit Thermal Lysis
Conclusion
TODAY’S PRESENTATION MOCK
Negative-sense ssRNA virus, Filoviridae family
Vector: fruit bats, nonhuman primates
Causes viral hemorrhagic fever Incubation Pd: 2-21 Days
Zaire, Sudan, Tai Forest , Bundibugyo strains cause disease in humans
Mortality rates: 50-90%; no current vaccine
Category A Bioterrorism Agent
EBOLA VIRUS (EBOV)
Distribution of EBOV cases in 2014 Outbreak. [WHO, Ebola Response Roadmap]
Filamentous Ebolavirus. 970 nm long; 80 nm in diameter. [Swiss Institute of Bioinfomatics,2014]
MOCK
EBOLA DIAGNOSTIC TOOLSType Target Source Detecti
on DayTest Time
Sensitivity
Remarks
Virus isolation
Viral particle Blood, tissue
2-7 1-2 weeks
unknown • Needs bio-containment (BSL-4) and temperature-controlled shipping
• High cost• Patient dead by time of
completionRT-PCR Viral nucleic
acidBlood, serum, tissue
2-7 < 2 hours
97% • Rapid; most sensitive method• Requires special equipment• Problem of cross-contamination• able to detect all serotypes with
degenerate primers• detects 24-48 hrs before Antigen
ELISAAntigenELISA
Viral antigen Blood, serum, tissue
3-7 > 3 hours
83% • Rapid and sensitive• Requires special equipment
Immunoassays
Virus-specific antibodies
serum > 6 days 1 day 67% • Prone to non-specific positives• Many patients are dead before
developing antibodies• Post-recovery or post-mortem
diagnostic“…the development and eventual use of RT-PCR for diagnosis of Ebola infection in African Laboratories is to be recommended.” –[Leroy et. al., Journal of Medical Virology]
MOCK
DEVICE DESIGN MOCK
IntroductionDevice design
Thermal Lysis of Ebola viral particles Off-chip thermal lysis
In water Optimization in plasma
On-chip photothermal lysisRT-PCR of Ebola viral particles
Comparison of Benchtop and Optical Cavity RT-PCR SpinColumn Kit Thermal Lysis
Conclusion
TODAY’S PRESENTATION MOCK
OFF-CHIP THERMAL LYSIS
0 5 10 15 20 25 3015
20
25
30
35
40
34.632.09
29.1227.65
26.12 25.98 25.76
Comparison of Thermal Lysis Durations for EBOV Particles
Thermal Lysis Duration (mins)
Ct V
alu
e
Control 40 50 60 70 8010
15
20
25
30
35 32.329.6
27.67 26.55 26.34 26
Comparison of Thermal Lysis Temperatures for EBOV Particles
Thermal Lysis Temperature (°C)
Ct V
alu
e
Temperature Comparison Conditions
Sample 105 RNA Particles/mL
Medium Water
Duration 20 minutes
Temperatures 40-80 °C
Duration Comparison Conditions
Sample 105 RNA Particles/mL
Medium Water
Duration 0-30 minutes
Temperatures 60 °C
MOCK
EFFECT OF PLASMA ON THERMAL LYSIS
0 10 20 30 50 9515
20
25
30
35
40
26.0127.28
29.931.2
35.09
38.2
Effect of Plasma on Thermal Lysis
% Plasma
Ct V
alu
e
Plasma Conditions
Sample 105 RNA Particles/mL
Medium Water + Plasma(0-95%)
Duration 20 minutes
Temperatures 60°C
MOCK
THERMAL LYSIS PERFORMANCE OFF-CHIP
Viral Load of Fatal Sudan Ebola Infections [Towner et. Al., 2004]
4 5 6 7 8 915
20
25
30R² = 0.985268878718535R² = 0.983140587989411R² = 0.983182869096114R² = 0.99957505577393
R² = 0.999513263567778
Comparison of Viral Particle Concen-trations in Off-Chip Thermal Lysis
SpinColumn Kit
Linear (SpinColumn Kit)
0% Plasma
Log RNA Particles/mL in RT-qPCR
Ct V
alu
e
Thermal Lysis Conditions
Duration 20 minutes
Temperature 60°C
MOCK
ON-CHIP PHOTOTHERMAL LYSIS
4 5 6 7 8 920
25
30R² = 0.984996993385448R² = 0.983140587989411
Off-Chip Thermal Lysis vs. On-ChipPhotothermal Lysis
Off-ChipLinear (Off-Chip)On ChipLinear (On Chip)Linear (On Chip)
Log RNA Particles/mL in RT-qPCR
Ct V
alu
eOn/Off Chip Comparison Conditions
Sample 104 -108 RNA Particles/mL
Medium 80% Water+20% Plasma
Duration 20 minutes
Temperature 60 °C
MOCK
IntroductionDevice design
Thermal Lysis of Ebola viral particles Off-chip thermal lysis
In water Optimization in plasma
On-chip photothermal lysisRT-PCR of Ebola viral particles
Comparison of Benchtop and Optical Cavity RT-PCR SpinColumn Kit Thermal Lysis
Conclusion
TODAY’S PRESENTATION MOCK
OPTICAL CAVITY PCR
[Son et. al., 2015]
MOCK
EXTREME RT-PCR: SPIN COLUMN KIT MOCK
Extreme RT-PCR Comparison Conditions
Sample 104 – 108 vp/mL
Medium Water
Lysis SpinColumn Kit
PCR Kit KAPA 2G Fast [extreme]
Cycling Conditions
95°C x 1 sec x 3560°C x 1 sec
RT-PCR: THERMAL LYSIS OFF CHIPMOCK
Extreme RT-PCR Comparison Conditions
Sample 104 – 108 vp/mL
Medium Water
Lysis Thermal, Off-Chip
PCR Kit KAPA 2G Fast [extreme]
Cycling Conditions
95°C x 1 sec x 3560°C x 1 sec
RT-PCR: THERMAL LYSIS ON-CHIP
Extreme RT-PCR Comparison Conditions
Sample 104 – 108 vp/mL
Medium Water
Lysis Thermal, On-Chip
PCR Kit KAPA 2G Fast [extreme]
Cycling Conditions
95°C x 1 sec x 3560°C x 1 sec
MOCK
This project has demonstrated the viability of photothermal lysis and optical cavity RT-PCR for Ebola diagnosis.
Future Goals:On-chip detection of
amplified productIntegration of thermal lysis
and PCR into one cavityApplication on patient
samples
CONCLUSION
Ebola Survivors. [The Telegraph (UK), 2015]
MOCK
