A lateral flow assay for quantitative detection of amplified HIV-1 RNA

Abstract

Although the accessibility of HIV treatment in developing nations has increased dramatically over the past decade, viral load testing to monitor the response of patients receiving therapy is often unavailable. Existing viral load technologies are often too expensive or resource-intensive for poor settings, and there is no appropriate HIV viral load test currently available at the point-of-care in low resource settings. Here, we present a lateral flow assay that employs gold nanoparticle probes and gold enhancement solution to detect amplified HIV RNA quantitatively. Preliminary results show that, when coupled with nucleic acid sequence based amplification (NASBA), this assay can detect concentrations of HIV RNA that match the clinically relevant range of viral loads found in HIV patients. The lateral flow test is inexpensive, simple and rapid to perform, and requires few resources. Our results suggest that the lateral flow assay may be integrated with amplification and sample preparation technologies to serve as an HIV viral load test for low-resource settings.

Figure 1. Lateral flow assay design.

The lateral flow assay is designed to detect a 142 bp amplified RNA sequence. The lateral flow strip consists of a conjugate pad containing gold nanoparticle probes (GNPs), a nitrocellulose membrane containing capture oligonucleotides, and an absorbent pad. Target RNA is dispensed onto the conjugate pad and binds to the GNPs. The target RNA – GNP complex flows down the strip and binds to the target capture sequence, while unbound GNPs bind to the positive control sequence. After wash buffer carries unbound GNPs down the strip, an enhancement solution is added to increase the optical absorbance of the captured GNPs. The signal of the GNPs captured in the detection zone should be proportional to the number of RNA copies dispensed onto the strip, providing quantitative detection.

Figure 2. Performance of the optimized lateral flow assay.

Lateral flow strips were made and tested on two different days (Batch1 and Batch2). The lateral flow assay was performed in duplicate (Batch1) or in triplicate (Batch2) using a dilution series of in vitro transcribed RNA. The number of RNA copies dispensed per strip ranged from 9.5 to 13 log₁₀ copies in steps of 0.5 log₁₀ copies. (A) Scanned image of one set of lateral flow strips. Note that although the contrast was adjusted in the figure, raw images were used for signal-to-background calculations. (B) Dose response curves based on the average signal-to-background ratio (SBR) for each log₁₀ copy number. The negative control SBR is shown for comparison. Error bars represent one standard deviation. The line and regression equation are shown to denote the linear range of the assay.

Figure 3. Performance of the lateral flow assay after storage.

To assess the effects of storage on LFA performance, lateral flow strips were fabricated on the same day, placed in foil pouches with desiccant, and stored at room temperature or 37°C. The lateral flow assay was performed and analyzed on the day of strip creation and 1, 3, 7, 14, 21, and 28 days after strip creation. The signal-to-background ratio (SBR) for each log₁₀ copy number is shown for strips performed on different days. The negative control SBR is shown for comparison. The regression line and equation were calculated for the average SBRs over the linear range of the assay, from 10.5 to 13 log₁₀ RNA copies. (A) Dose response curves at each time point and (B) average dose response curve for strips stored at room temperature. (C) Dose response curves at each time point and (D) average dose response curve for strips stored at 37°C.

Figure 4. Detection of NASBA products.

NASBA was performed using 0, 5, 50, 500, or 5000 copies of in vitro transcribed HIV gag RNA as a template. Products were diluted by a factor of 10, 100, and 1000, and a 20 µL volume of each dilution was dispensed onto a lateral flow strip for detection. (A) Scanned image of strips for each concentration of NASBA products and dilution factor. Note that although the contrast was adjusted in the figure, raw images were used for signal-to-background calculations. (B) Dose response curve constructed using the signal-to-background ratio of 100-fold diluted products. The negative control SBR is shown for comparison.