Vortex- and Centrifugation-Free Extraction of HIV-1 RNA

Abstract

Background and objective: HIV viral load measurements play a critical role in monitoring disease progression in those who are on antiretroviral treatment. In order to obtain an accurate measurement, rapid sample preparation techniques are required. There is an unmet need for HIV extraction instruments in resource-limited settings, where HIV prevalence is high. Therefore, the objective of our study was to develop a three-dimensional (3D) microfluidic system to extract HIV-1 RNA with minimal electricity and without complex laboratory instruments.

Methods: A 3D microfluidic system was designed in which magnetic beads bound with nucleic acids move through immiscible oil-water interfaces to separate HIV-1 RNA from the sample. Polymerase chain reaction (PCR) amplification was used to quantify the total amount of HIV-1 RNA extracted as we optimized the system through chip design, bead type, carry-over volume, carrier RNA concentration, and elution buffer temperature. Additionally, the extraction efficiency of the 3D microfluidic system was evaluated by comparing with a Qiagen EZ1 Advanced XL instrument using 20 HIV-1-positive plasma samples.

Results: Our method has near-perfect (100%) extraction efficiency in spiked serum samples with as little as 50 copies/mL starting sample. Furthermore, we report carry-over volumes of 0.31% ± 0.006% of total sample volume. Using the EZ1 Advanced XL as a gold standard, the average percentage HIV-1 RNA extracted using the microchip was observed to be 65.4% ± 24.6%.

Conclusions: From a clinical perspective, the success of our method opens up its possible use in diagnostic tests for HIV in the remote areas where access to vortexes and centrifuges is not available. Here we present a proof-of-concept device which, with further development, could be used for sample preparation at the point of care.

Fig. 1

The microfluidic chip used in the experiments. a Side of chip showing dimensions of reservoirs and channels. The oil channel is 1 mm high and 2 mm wide, as shown in the cross-section. b The sample well is shown in blue, the oil channel in pink, and the elution well in green

Fig. 2

Forces acting on the bead aggregate at the sample oil interface. $F_D$ hydrodynamic drag force, $F_I$ interfacial rtension force, $F_M$ magnetic driving force

Fig. 3

Elution buffer temperature versus the percentage HIV RNA recovered

Fig. 4

Percentage HIV RNA extracted at varying carrier RNA volumes

Fig. 5

a Relative fluorescence units versus cycle number for polymerase chain reaction (PCR) plots for serially diluted 1 mL serum samples from 540,000 to 54 copies/mL. Additionally, a no template control and a positive control extracted using spin column kit were included in the run. b Cycle threshold versus the log copy number of HIV spiked into serum samples. c Input versus extracted HIV copy number with an R² value of 0.9995. The extracted copy number was extrapolated from the best fit line in b. Adj. adjusted, Ct cycle threshold, NTC no template control

Fig. 6

Viral load versus percentage of HIV RNA recovered using the microfluidic chip. Matched samples with the Qiagen EZ1 Advanced XL instrument were assumed at 100% extraction efficiency