Download Rapid Point of Care Molecular Diagnostic Test for Ebola

Rapid Point of Care Molecular Diagnostic Test for Ebola Virus
Jason Benzine,1 Dipankar Manna,1 Chad Mire,2 Thomas Geisbert,2 Eric Bergeron,3 David Mead,1 and Yogesh
1
Chander
1Lucigen
Corporation, Middleton, WI 53562; 2University of Texas Medical Branch, Galveston, TX; 3Centers for
Disease Control and Prevention, Atlanta, GA
D) Instrumentation
A) Isothermal Detection of EBoV
• Required instrumentation: Heat block (for heat lysis) and a
Isothermal block (AmpliFire™, Douglas Scientific, MN).
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LAMP primers were designed targeting a conserved region of the spike
glycoprotein (GP) gene.
Amplification was performed using OmniAmp™ DNA polymerase.
Target: Serial dilutions of genomic RNA of two EBoV strains: Kikwit
(1995) and Guinea (2014).
Reaction temperature optimal of 72°C was obtained by thermal cycler
gradient profile.
Real time monitoring of amplification by using DNA intercalating dye in
the amplification mixture.
Assay time: 40 min.
Table 1: Sensitivity of EBoV LAMP assay
Zaire – Kikwit (1995)
This assay is based on Loop-mediated isothermal AMPlification
(LAMP) of the RNA viral genome of the current EBoV-Zaire strain.
Analytical sensitivity was determined by testing 10-fold serial
dilutions of RNA extracted from Ebola virus – Zaire, Kikwit (1995)
strain and a strain from the ongoing outbreak (Guinea-2014) in W.
Africa. A true POC test for Ebola was developed, consisting of
lyophilized amplification reagents, a rapid one step sample
preparation method, and a battery operated isothermal instrument
capable of detecting a fluorescent signal in positive reactions.
108
7 x
7 x 107
7 x 106
7 x 105
1.4 x 105
7 x 104
7 x 103
NTC
•
Marker
0.46
0.18
1.18
0.10
1.13
0.33
4.41
4.46
2.8 x
2.8 x 107
2.8 x 106
2.8 x 105
5.6 x 104
5.6 x 103
2.8 x 103
NTC
11.0
11.7
13.2
15.6
18.4
25.5
27.4
48.7
0.1
0.3
0.3
0.9
1.4
4.4
3.5
4.5
EBoV
MAR
LF-Josiah LF-Sauer CCHFv
WNV
CHIKV
Figure 3: AmpliFire™
E) Workflow for POC EBoV LAMP test:
DENV-1 DENV-2 DENV-3 DENV-4
Conclusions
Figure 1: Specificity of EBoV LAMP assay.
• EBoV LAMP diagnostic test with results
C) EBoV LAMP using dried reagents
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available in under 40 minutes.
• High sensitivity (2800 cp/rxn) for current
outbreak strain.
• High specificity, no amplification of any other
viral targets.
• Simple, rapid sample preparation step (5 min.
heat lysis).
• Dried reagents for room temperature storage.
• Simple and easy to use workflow.
• Suitable for POC use, no need of extensive
40
30
20
training or expensive instrumentation.
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• Further work is underway to develop the test
EBoV - Kikwit (1995)
EBoV - Guinea (2014)
Target Copies/test
Figure 2: Sensitivity of EBoV LAMP using dried reagents.
NTC
NTC
8750
17500
35000
700000
NTC
NTC
21875
0
43750
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87500
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175000
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Amplification reagents were lyophilized to ensure long term stability of
LAMP reagents for use in field conditions without maintaining cold
chain.
Complete formulation (master mix, LAMP primers, and dye) were
added to 0.2 ml PCR tubes, and dried overnight in a lyophilizer.
After lyophilization, tubes were stored at room temperature in a pouch
with a desiccant.
To test performance of lyophilized reagents, 25 ml of 10-fold dilution of
target was added to dried reagents and tubes were incubated at 72°C.
350000
•
1750000
Here we report on development of a simple, sensitive, and easy to
use diagnostic test to allow rapid detection of EBoV at POC. This test
is based on LAMP and carried out on a commercially available
isothermal instrument (AmpliFire™, Douglas Scientific, MN) to allow
real time detection. This instrument is light weight (< 5 lbs.), highly
portable, small foot-print, and battery operated. Results of assay are
available in under 1 hr., allowing multiple samples to be tested in a
short time. Furthermore, to ensure long term stability of reagents,
amplification reagents were freeze dried, without any loss in
efficiency.
16.6
14.5
16.5
19.4
25.5
26.9
30.4
48.70
108
Specificity was determined by testing RNA extracts from closely related
viruses: Lassa Fever virus (LV)- Josiah strain, Marburg virus (MARV)Musoke strain, Chikungunya virus (CHIKV), Crimean–Congo
hemorrhagic fever virus (CCHFV), West Nile virus (WNV), and Dengue
fever virus (DENV) serotypes 1 – 4.
Time to Result (min)
A rapid and simple diagnostic test which can be used at point-of-care
without much training is needed. Such a test will allow patient
samples to be verified at local clinics with results available in a few
hours, thus helping in interruption of virus transmission in severely
resource-constrained settings. Having such a test will enable health
services to better manage not only current Ebola outbreak but also
better prepare for a possible recurrence of Ebola in W. Africa as well
any where else.
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Light weight (< 5 lbs.) and portable
Small foot-print and battery operated
8- wells
Touch-screen user interface for easy operation,
Real time monitoring of amplification reaction
Results are displayed on the screen as “positive” or
“negative”
Data can be stored on the device itself or transferred using
a USB port.
B) Assay specificity
In the absence of any effective vaccine or antiviral therapy, the best
way to control spread of Ebola virus (EBoV) is to interrupt the
transmission cycle. This requires availability of diagnostic tests to
rapidly confirm or discard suspected cases to help in clinical
decisions and to facilitate early detection of cases.
During the current Ebola outbreak in West Africa, efforts to break the
transmission cycle was hampered by cumbersome, slow and
complex diagnostic tests. Standard molecular assays such as RTPCR, though highly specific and sensitive, require trained laboratory
staff to run the assay which involve a number of laborious steps. Also,
running these assay’s requires a full tube of blood, transportation of
sample to a central location takes from 2 to 6 hours, and costs around
$100. These requirements are difficult to meet in resourceconstrained West African settings, thus severely limiting testing
capacity. Delayed testing means infected people remain in the
community with a severe risk of unknowingly transmitting the virus to
others.
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RNA copies
Time to
RNA copies
Time to
Std. Dev.
Std. Dev.
per rxn.
Result (min.)
per rxn.
Result (min.)
Results obtained show that this simple and easy to use diagnostic
test can be used for rapid detection of both strains of EBoV with
high sensitivity and specificity and sensitivity in 40 minutes at POC.
Background
• Features of AmpliFire™
Zaire – Guinea (2014)
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Nucleic acid testing (NAT) methods are now considered to be gold
standard for detection of pathogens. These methods allow higher
sensitivity and specificity compared to alternatives based on
antibodies and culturing. The requirement of expensive equipment,
specialized laboratories, and trained personnel precludes routine
NAT in most of the developing world and even in resource limited
parts of the United States. The ongoing outbreak of Ebola virus
(EBoV) in West Africa has highlighted the need for rapid molecular
diagnostic tests which can be used at point-of-care (POC) with
limited training and simple equipment. Here we report on the
development of a rapid, sensitive, low cost but facile NAT method
for robust detection of EBoV at POC.
Results
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Abstract
and submit for Emergency Use Authorization in
2015.
Acknowledgement
This work was supported by grant from NIAID to Dr.
Yogesh Chander.