Development of a Point of Care Assay for Detecting High Risk HPV in Resource Limited Settings
More than 85% of women who die of cervical cancer live in low- and middle- income countries. The main reason for this disparity stems not from lack of methods for combating cervical cancer, but rather their inaccessibility in resource limited areas. Over 90% of cervical cancer develops from a persistent infection with a high risk type of human papilloma virus (HPV); early detection and treatment of high risk Human papilloma virus subtypes can prevent cervical cancer fatalities. However, detection is expensive, time consuming, and requires specialized equipment. Therefore to prevent cervical cancer deaths, an inexpensive and easy-to-use assay must be developed that can detect high risk HPV at the point of care. This project is to develop a high-throughput screening assay for four of the most common high risk HPV types directly from patient samples using an integrated portable device based on lens-free imaging. The device can be created using a 10x10mm glass coverslip coated with PDMS and etched to create multiple lanes. Surface functionalization chemistry will be used to attach DNA probes for four HPV types, one in each lane. DNA from patients’ cervical brushings will be captured by complementary DNA probes. Magnetic microbeads conjugated to DNA probes will be attracted towards the functionalized surface by use of a magnet, and then magnetically washed away. Target DNA will capture the magnetic beads with the corresponding probe, and these beads will remain specifically bound to HPV-specific DNA while other beads are washed away. Using the Digital Diffraction Diagnostic (D3) lens free imaging device previously developed and optimized at MGH, the beads in each lane will be counted for accurate diagnoses. This device will be further optimized to diagnose multiple patients simultaneously to increase throughput. We will also optimize strategies for DNA amplification to enhance use in resource limited settings. Amplification by PCR requiring a thermocycler will be replaced by an isothermal amplification known as Recombinase Polymerase Amplification (RPA). In summary, cervical cancer mortality can be prevented by the detection and treatment of high risk HPV, and the vision of this project is to make that possible across the world.
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