Pakistan-US Science and Technology Cooperation Program |
Phase 7 (2017 Deadline)
Health security: Point of care, multiplexed molecular detection of infectious diseases endemic in Pakistan
US Partner: Haim Bau, University of Pennsylvania
Pakistan Partner: Muhammad Shabir, International Islamic University
This project will train Pakistani researchers in the use and implementation of a newly developed platform for the detection of hepatitis C, malaria, and HIV in blood samples, pathogens endemic in Pakistan and associated with significant morbidity, mortality, and socio-economic cost. The platform will also be expandable for use with other targets and samples, including environmental and agricultural samples and food products.
2018: Our team has developed a two stage isothermal enzymatic amplification assay for the detection of nucleic acids associated with the human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The analytical performance of this assay was tested at Penn and patient samples were tested in Pakistan (single-plex) for HIV and HCV on benchtop equipment. Patients’ test results obtained with the Penn isothermal assay were critically compared with standard quantitative PCR tests (the gold standard).
A 3D-printed microfluidic chip was designed, fabricated, and tested. The chip accommodates multiple reaction chambers, each for a single-plex assay. Each reaction chamber is equipped with a nucleic acid isolation membrane at its inlet. When lysed samples filter through the isolation membranes, nucleic acids bind to the membranes while the rest of the sample is discharged to waste. Our isolation membrane allows one to decouple sample volume from reaction chamber volume, allowing higher sensitivity than common in rapid molecular tests. We incubate the chip with a simple custom-made processor that maintains the chip at a desired temperature for the duration of the test. Nucleic acid amplification is monitored by detecting emission intensity from either intercalating fluorescent dye or luciferous dye that emits light without excitation with a USB-based microscope or the ubiquitously available smartphone camera. The operation of the chip requires manual pipetting of wash solutions.
To minimize manual operations, the team is developing a slider cassette that will house all wash solutions and wash. The slider cassette consists of slider housing the nucleic acid isolation membrane and reaction chamber and a house hosting blisters with wash solutions and absorption pads. We have demonstrated that the slider cassette successfully incubates enzymatic amplification. The development of the cassette has not been completed, however, and will continue in the next grant period.