molecular test to detect dengue, zika & chikungunya … · showing standard curves generated...
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© 2015
Molecular test to detect Dengue, Zika & Chikungunya
and run on the QuRapID LV platform.
© 2017
Dr David Edge BioGene
Dr Emily Adams LSTM
03/05/17
© 2015
• Technology was initially developed for the rapid detection of sepsis
causing bacteria.
• Adapted in 2015/2016 for the detection of Ebola- the QuRapID
technology demonstrator for the EbolaCheck project.
© 2017
© 2015© 2017
Add 450ul of
extraction
buffer by fixed
volume pipette.
Close reaction vessel cap
and place into instrument
for extraction step-vessel is
subjected to 4 cycles of -18
to 40C followed by 5 seconds
at 700C
5 min Reverse
Transcription step
30 min Q-PCR step
<50 mins
Simplified results displayed indicating
which pathogen is detected
Add 25ul of
whole blood
© 2015
Extraction is performed in tube by rapid cyclical freezing and thawing of the sample in the
actual RT-PCR mastermix.
• Freezing the sample introduces mechanical damage from ice formation.
• Thawing the frozen sample generates osmotic shock as water flows rapidly back into
the cell.
Direct processing of
blood, no lab access
required-simply add
fingerprick of blood into
PCR reaction vessel.
© 2017
© 2015
1. Inhibition of amplification
2. Inhibition of fluorescence signals
Spectrum resulting from SYBR green QPCR with (right) and without (left) human blood at 10%-95% inhibition of fluorescence
Technical limitations to blood in QPCR.
Identical RT-QPCRs at 0% and 6% whole
human blood using standards reagents and
instrumentation-signal/amplification
suppressed over 95%
© 2017
© 2015
Optics for QPCR detection in the presence of up to 20% whole blood
High powered laser spectrophotometry-makes
possible triplex detection of fluorophores in the
presence of up to 20% blood.
Real-time RT-QPCR in the presence of 12% whole human
blood- 1 spectrum captured for each of the 45 PCR cycles.
© 2017
© 2015
Mastermix-optimised for consistent amplification at up to 12%
whole blood added directly into the PCR. Has been tested for a
range of matrices including swabs, sputum.
Mastermix reliably amplifies at up to 12% whole bloodOptimised for 8% blood
© 2017
© 2015
Detection of Ebola Zaire direct from whole human blood (in
publication)
Three replicates of armoured RNA surrogate containing EBV GP/NP inserts at 100-1000000 molecules
per reaction and at 8% whole blood.
© 2017
© 2015© 2016
Figure
2.
Figure
1.
Figure 1. Showing A) efficiency of lysis for an enveloped virus, expressed as number
of cycles against Ct value. B) Performance of the dedicated one step RT-QPCR
mastermix in the presence of varying percentages of whole blood-the Ebolacheck
assay was optimised for 8% whole blood. C) Detection of 66 viral genome equivalents
contained in 5ul of whole blood (8% of final reaction volume).
Figure 2. Showing standard curves generated for live Ebola virus detected in the 62.5µl
closed tube Ebolacheck assay. A) The 1976 Ebola strain spiked into reactions at 3 serial
dilutions and plotted as plaque forming units of the original viral culture against detection
Ct B) a plot of genome equivalents per reaction against Ct for two Ebola strains and an
internal control armoured RNA. The graphs demonstrate the linearity of the detection
methodology.
© 2015
• A rapid technology-multiplexed detection of viruses from whole blood~50 minutes. Working on
assays for Zika, Chikungunya, Dengue serotyping, Ebola, Lassa, Malaria and others.
• In-field point of care diagnostics.
• No requirement for laboratory facilities, cold chain or expert users
• Automated analysis and random access.
• Reduced costs per test by removing time and effort of RNA extraction.
• Ability to be adapted easily to alternate matrices and targets.
• Extraction method is completely linear with a LLOD of <4,000 genome equivalents/ml blood.
© 2017
Summary:
The only current molecular
approach that can detect multiple
RNA viruses from a single whole
blood sample.
© 2015
ChikZika
Den1 Den3Assays for
1. Ebola, Lassa, Marburg, Rift valley
fever, Crimean Congo.
2. Dengue 1-4 serotyping, Zika,
Chikungunya.
© 2017
6 samples at
5.35x107/ml
NTC
© 2015© 2017
Wider ideas
• Assay will render virus non-infectious.
• Human RNA markers are also in the blood sample.
• Works on a range of matrices and targets.
ThanksDFID/Save the Children/Wellcome trust-Ebolacheck
DOH QuRapID LV
Innovate UK for funding the background IP
PHE- Dr Kevin Richards, Dr Miles Carroll
Dr Sterghios Moschos
LSTM
© 2015
• In 2015 LSTM and BioGene were awarded a MRC Zika Emergency fund project. • Design of direct from blood molecular tests for Zika, Dengue (typed) and
Chikungunya in the Americas. • Prospective evaluations in Brazil and Guatemala• Response to other viral/bacterial/parasitological infections with design of new
probes as required.
© 2015
Probe design and field collection and testing • Visual OMP used for probe design and optimisation, able to visualise secondary
structure of target region
• Testing on live viral culture in LSTM’s Category 3 laboratories, including Dengue serotypes, Zika and Chikungunya (to come)
• Prospective collection of patient samples in Guatemala and Brazil – field testing commencing in July 2017 on whole blood. Comparison with reference standard molecular tests in referral laboratory
• Sample collection to be created and happy to discuss sample types and access
© 2015
What would speed up the process of product development, validation and distribution
• Development and supply of cheap enzyme in order that large volume reactions can be used.
• Funding larger field collections in Guatemala for storage, testing and distribution