| EPSRC Reference: |
EP/K503629/1 |
| Title: |
Rapid, on-chip, multiplexed detection of sepsis-causing organisms from blood samples |
| Principal Investigator: |
Greenman, Professor J |
| Other Investigators: |
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| Researcher Co-Investigators: |
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| Project Partners: |
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| Department: |
Clinical Biosciences Institute |
| Organisation: |
University of Hull |
| Scheme: |
Technology Programme |
| Starts: |
01 October 2012 |
Ends: |
30 June 2016 |
Value (£): |
64,620
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| EPSRC Research Topic Classifications: |
| Med.Instrument.Device& Equip. |
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| EPSRC Industrial Sector Classifications: |
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| Related Grants: |
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| Panel History: |
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Summary on Grant Application Form |
This proposal brings together BioGene's expertise in development of rapid PCR, instrument design & manufacture and
software development, with the University of Hull's proven track-record in microfluidic chip design & optimisation, and
fabrication of devices with real world interfaces, e.g. scene of crime or point of care. Together the group will build a rapid
and portable, highly-accurate, system capable of extracting pathogen DNA from a blood sample, to determine the presence
of multiple bacterial species with relevant sub-type specificity, e.g. coagulase status for Staphylococci. All reagents for DNA
extraction, PCR amplification and separation will be preloaded in the device. The operation and stability of this instrument
are based on published work from the Hull team.
BioGene have a proven optical system capable of separating more than 10 dyes, and when this technology is coupled with
the ability to electrophoretically separate PCR products down to 2 base pair resolution, using Hull's microfluidic device, the
new instrument will be able to discriminate 1000s of distinct PCR fragments. The detection system is based on optical
deconvolution of the fluorescence signal generated by the multitude of differentially-labelled products. This method enables
the detection of many more targets than is currently possible with conventional sequencers. In this proof of concept study
we intend to characterise at least 10 specific factors, i.e. 8 organisms and two species-specific factors; it is anticipated
however that a considerably greater level of multiplexing will be achieved.
Blood is widely recognised as a "difficult medium" for pathogen nucleic acid amplification, both due to the presence of a
wide range of PCR inhibitors and the vast excess of human genomic DNA present. The blood sample will first be treated to
lyse red blood cells and facilitate collection of bacterial cells, initially by filtration, followed by recovery of the concentrated
cells to provide the starting material for the DNA analysis. The aim is to produce a platform suitable for handling microlitre
volumes as this would allow detection of sepsis in neonates and children where blood collection is via capillary tubes. An
additional benefit of using smaller volumes of blood is a reduction in complexity and cost of the overall unit.
The focus of the PCR system will be to maximise sensitivity and speed. Optimal PCR primer pairs for each target will be
identified after extensive in silico modeling before being tested in a real-time PCR format and for compatibility in a multiplex
mix. A selection of the most common organisms will be chosen from a list of sepsis-cuasing organisms in Hull & East
Yorkshire patients during 2010. Other key factors that will be addressed to deliver the system are: an effective sample
interface and automation of DNA extraction; incorporation of the ultra-rapid PCR unit and optical system onto a microfluidic
platform; and bespoke control software that includes data storage and user-friendly operator display.
Each part of the system will be tested extensively during design of the assays themselves as well as construction of the
proof of concept unit using blood samples from donors spiked with genome equivalents of the targets in order to test
functionality. Testing of the fully-functional device will be completed on blood drawn from patients: i) newly-presenting at
Accident & Emergency with classic symptoms of sepsis (n=50) or ii) on the Intensive Care Unit (n=15). The pathology
reports, obtained for clinical assessment, will give a "gold-standard" for comparison. Dr W Townend (Emergency Medicine),
Dr S Bennett (Cardiothoracic Anaesthesia and Intensive Care) and Dr R Meigh (Microbiologist), all Consultants at Hull &
East Yorkshire NHS Trust, will act as a clinical advisory team. Appropriate certification, eg. CE marking and IVD
certification (98/79/EC), will be applied for once the clinical trials have been compl
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Key Findings |
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This information can now be found on Gateway to Research (GtR) http://gtr.rcuk.ac.uk
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Potential use in non-academic contexts |
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This information can now be found on Gateway to Research (GtR) http://gtr.rcuk.ac.uk
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Impacts |
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| Description |
This information can now be found on Gateway to Research (GtR) http://gtr.rcuk.ac.uk |
| Summary |
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| Date Materialised |
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Sectors submitted by the Researcher |
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This information can now be found on Gateway to Research (GtR) http://gtr.rcuk.ac.uk
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| Project URL: |
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| Further Information: |
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| Organisation Website: |
http://www.hull.ac.uk |