© 2015

Molecular test to detect Dengue, Zika & Chikungunya

and run on the QuRapID LV platform.

© 2017

Dr David Edge BioGene

(david@biogene.com)

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

© 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

Co-infection of DENV and CHIKV present in 32% of positive samples (2015)

46

CHIKV DENV

© 2015

• Sensitivity required?

• Low viraemia in acute Zika infection

© 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

© 2015

• Thomas Edwards

• Prof Luis Cuevas

• Chris Williams

• Kavit Shah

• Adam Tyler

• Nelson Nazareth

• Leticia del Carmen Castillo Signor - Guatemala

• Eunis Donis

• Ricardo Gurgel - Sergipe