KESS2 East PhD in Rapid Pathogen Detection: Universal sample preparation platform for rapid diagnosis of infection

Outline

This PhD studentship will focus on the development of a rapid microwave based system to extract DNA from clinical samples.

Background

In order to make biomarkers available for detection, target DNA must be extracted from the clinical sample. Sputum is a complex mixture of organic material which contains a mixture of mucus, food debris and lung derived cells some of which contain bacteria trapped within.

The distribution of these bacteria in the sample is uneven, making it challenging to detect the presence of target DNA. Indeed, increasing the amount of sample processed increases the chance of detecting the biomarker.

Project aims and methods

To address this issue, we will develop a sample processing cartridge which will employ ultrasound to homogenize the sample, followed by exposure to pulsed microwaves of around 10 W rms power to release the DNA biomarkers from bacteria containing cells into the surrounding medium.

In this part of the extraction process, the duty cycle of the pulsed microwaves will be used to prevent heating, which might otherwise denature the target DNA. The released single stranded DNA will then be captured by specific DNA probes chemically linked to the surface of magnetite beads (a streptavidin/biotin system).

An electromagnet will then be used to recover the beads and their DNA cargo from the organic material. Following washing to remove extraneous organic material, the retained beads will be heated to 60°C to cause detachment of the target DNA. This detachment could be performed via directed microwave heating, or use conventional heating methods.

To achieve the objective, we have identified the following three work packages (WP):

• WP1. Design the sample processing  cartridge and create prototypes
• WP2. Identify the nanoparticle DNA probe combinations which maximise the recovery of target DNA from microwaved samples.
• WP3. Identify the cartridge/ nanoparticle combination which maximises target DNA recovery in a form compatible with a lateral flow assay

The outputs from WP1 and WP2 will be combined to identify the approach which maximises the recovery of target DNA. This will be determined using simulated clinical samples spiked with known concentrations of Mycobacteria. The level of sensitivity achieved will be compared to that seen using stand microscopy based screening (ZN).

The industrial partner Microchip will support the activities in WP 1 and WP2 ensuring manufacturability and suitability for volume assembly. They will also support the design and assembly of microwave control and actuation.

What is funded

For more information on what is funded, please see our website: https://www.cardiff.ac.uk/study/postgraduate/funding/view/kess2-east-phd-in-rapid-pathogen-detection-the-development-of-a-rapid-microwave-based-system-to-extract-dna-from-clinical-samples

Duration

3 years

Eligibility

For eligibility criteria, please see our website: https://www.cardiff.ac.uk/study/postgraduate/funding/view/kess2-east-phd-in-rapid-pathogen-detection-the-development-of-a-rapid-microwave-based-system-to-extract-dna-from-clinical-samples