| Viral load testing using polymerase chain reaction (PCR) is the preferred method for early detection of HIV-1 therapeutic failure. However, it is not widely available in resource limited settings due to the complexity of sample preparation, especially nucleic acid (NA) purification and the need for costly fluid handling systems. A novel NA extraction and purification method was developed that replaces multiple wash steps with a single passage of paramagnetic particles (PMPs) though an immiscible phase filter (IPF). Only two aqueous solutions are required: a lysis buffer, in which NAs are captured on PMPs, and an elution buffer where they are released for amplification. A channel containing liquid wax connects the lysis chamber to the elution chamber. Transporting PMPs through the immiscible phase yielded DNA and RNA as pure as that obtained after extensive wash steps required by comparable purification methods. The versatility of the purification method was 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. A disposable microfluidic cartridge was developed for the IPF method where 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. The liquids were released into the cartridge chambers by bursting the blisters' heat seals with motor-driven plungers. This allows for fluid storage without a cold chain and eliminates expensive fluid handling systems. Disposable modules were also developed to collect blood from a finger stick and passively separate plasma from whole blood using filter membranes. This modules act as a measuring device for plasma volume, eliminates fluid pipetting and integrates with the IPF cartridge. Therefore, we have demonstrated the feasibility of creating a low-cost platform for viral load measurement with few simple user steps. |
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