Technologies for Disease Management

HIV-1 Viral RNA Assay
Viral load monitoring using quantitative PCR (qPCR) is the only molecular tool that allows early detection of HIV-1 treatment failure. While quantitative PCR (qPCR) has transformed detection of nucleic acids (NAs) into a simple, closed-tube process; it remains a difficult procedure with complex biological samples because extensive purification is required to first remove inhibitors which affect reaction efficiency and the accuracy of results. No point-of-care viral load monitoring tool exists. Creating such a device entails integrating: 1) Blood collection and plasma separation 2) NA extraction 3) qPCR consisting of thermal cycling and optical measurement 4) reagent storage.

  HIV-1 Viral RNA Assay

We have developed a low-cost sample collection module to collect blood from a finger stick to eliminate the use of extensive vacuutainers and a trained phlebotomist to collect blood. Viral load testing requires the use of a centrifuge to separate plasma from whole blood. Our collection module passively separates plasma from whole blood using membranes, eliminating the centrifuge and also readily integrates into our cartridge for viral load measurements.

To integrate nucleic acid (NA) extraction and isolation with quantitative PCR (qPCR) amplification and detection,  a two-chamber cartridge was developed with an immiscible fluid barrier (IPB) separating the aqueous solutions in the sample and PCR chambers. NAs were adsorbed onto paramagnetic particles (PMPs) in the sample chamber and magnetically transported though the IPB directly into the PCR chamber where they were released. This eliminates multiple wash steps and enables the cartridge to be sealed throughout processing.  During cartridge fabrication, the lysis, elution, and immiscible fluids were dispensed into foil-laminate blisters, heat sealed, then bonded onto the cartridge over ports connected to the internal chambers.  After plasma was added to the sample chamber, the liquids were released into the cartridge chambers by bursting the blisters heat seals with motor-driven plungers. The versatility of the purification method has previously been demonstrated with blood, plasma, and urine in a laboratory configuration with quantitative detection of HIV-1 viral RNA, chlamydia, gonorrhea, and proviral HIV-1 DNA.  Preliminary results demonstrated quantitative detection of HIV-1 RNA from plasma.

The IPB cartridge can be readily integrated with a thermal cycler and fluorimeter. The fluorimeter and thermal cycler sub-systems are currently under development. An integrated circuit board is being designed to integrate all automated processes, thereby allowing the device to work as a stand-alone system without the use of a computer.

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