Abstract
While identifying acute HIV infection is critical to providing prompt treatment to HIV-positive individuals and preventing transmission, existing laboratory-based testing methods are too complex to perform at the point of care. Specifically, molecular techniques can detect HIV RNA within 8–10 days of transmission but require laboratory infrastructure for cold-chain reagent storage and extensive sample preparation performed by trained personnel. Here, we demonstrate our point-of-care microfluidic rapid and autonomous analysis device (microRAAD) that automatically detects HIV RNA from whole blood. Inside microRAAD, we incorporate vitrified amplification reagents, thermally-actuated valves for fluidic control, and a temperature control circuit for low-power heating. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) products are visualized using a lateral flow immunoassay (LFIA), resulting in an assay limit of detection of 100 HIV-1 RNA copies when performed as a standard tube reaction. Even after three weeks of room-temperature reagent storage, microRAAD automatically isolates the virus from whole blood, amplifies HIV-1 RNA, and transports amplification products to the internal LFIA, detecting as few as 3 × 105 HIV-1 viral particles, or 2.3 × 107 virus copies per mL of whole blood, within 90 minutes. This integrated microRAAD is a low-cost and portable platform to enable automated detection of HIV and other pathogens at the point of care.
Highlights
Despite the effectiveness of antiretroviral therapy (ART) to suppress viral loads and decrease HIV-related mortality, HIV remains a global epidemic
The optimal Reverse transcription loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) assay temperature for these primers is 65 °C as this temperature yielded the strongest test band intensity on lateral flow immunoassay (LFIA) illustrated in Fig. S5.† To assess specificity of the reverse transcription (RT)-LAMP assay, primers were tested with RNA from dengue virus (DENV) and chikungunya virus (CHIKV)
As predicted by LAMP restriction digest fragment analysis,[29] digestion with either of these enzymes resulted in smaller fragments compared to the undigested product, with the PstI digested product collapsing to the shortest fragment seen on the agarose gel in Fig. S7.† Taken together, these experiments suggest that the designed primer set targets the intended region in the gag gene and that this target amplification is specific to HIV-1
Summary
Despite the effectiveness of antiretroviral therapy (ART) to suppress viral loads and decrease HIV-related mortality, HIV remains a global epidemic. The dried RT-LAMP reagents were rehydrated with buffer (Table S3†) and virus (positive samples) or DEPC water (negative controls) in tubes or in the polyether sulfone (PES) amplification zone within the integrated device.
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