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Details of Grant 

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:
haswell, Professor s Dyer, Dr CE
Researcher Co-Investigators:
Project Partners:
Department: Clinical Biosciences Institute
Organisation: University of Hull
Scheme: Technology Programme
Starts: 01 October 2012 Ends: 30 June 2016 Value (£): 64,620
EPSRC Research Topic Classifications:
Med.Instrument.Device& Equip.
EPSRC Industrial Sector Classifications:
Related Grants:
Panel History:  
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
Key Findings
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Potential use in non-academic contexts
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Date Materialised
Sectors submitted by the Researcher
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Project URL:  
Further Information:  
Organisation Website: http://www.hull.ac.uk