(H-288) Amplicon-contained lateral flow test that enables sequential delivery: a demonstration on HIV drug resistance detection

Introduction:: Human immunodeficiency virus drug resistance (HIVDR) undermines the effectiveness of antiretroviral treatment (ART). Thus, HIVDR testing is important for treatment management. Previously, the laboratory-based oligonucleotide ligation assay (OLA) was developed for HIVDR detection in which HIV DNA was tested for its presence of mutations conferring HIVDR using allele-specific probe ligation and visualization of ligated probes via enzyme-linked immunoassay (ELISA) [1]. To speed up assay time, the ELISA step was simplified into a lateral flow test (LFT) to enable visual detection and avoid the need for a plate reader and a plate washer [2]. However, the use of conventional LFTs in molecular assays like OLA involved opening tubes containing amplicon and several transfer steps to enable (i) displacement or de-hybridization of the unligated probes from its mismatched HIV templates to eliminate false mutant signal (Fig. 1a) (ii) sequential delivery of ligation product and detection buffer on the LFT. These steps expose substantial risks for amplicon contamination and increase the complexity of LFTs, especially when users are less experienced with molecular diagnostics. Here we present a streamlined workflow that enables sequential delivery of ligation product and detection buffer without “opening” amplicon-containing tubes. This custom self-contained LFT has the potential to improve the implementation of OLA for HIVDR in low-resource settings by improving ease of use and reducing potential amplicon contamination in laboratories.

Materials and Methods:: We developed a custom LFT device prototype (Fig. 1b) with two sample ports with plastic spikes, reaction tubes with custom foil-sealed caps, and a reading window at the bottom to enable data collection by a flatbed scanner. Sample tubes are loaded into the sample ports, and the foil-sealed caps are punctured to release the two fluids, establishing containment within the LFT. To evaluate the flow, tubes with two different colored fluids were tested, and the reading window was scanned over 20 minutes. Next, we tested the sequential delivery function of our LFT device. The sample tube contained HIV wild-type (WT) templates annealed to their mismatched mutant (MUT) allele-specific and common probes, representing “unligated” samples that can cause false mutant signals. The detection tube contained gold solution either with or without competing oligonucleotides, which upon successful delivery, can displace the unligated probes and eliminate false mutant signal. Both sample and detection tubes were added to the sample ports and punctured sequentially. The scanned images were taken after 20 minutes and analyzed using a Python code that outputted values for the intensity of visible bands.

Results, Conclusions, and Discussions:: Our flow experiment showed successful sequential delivery of two color fluids (Fig. 1c), switching from blue (first sample tube) to red (second sample tube) across a 20-minute detection time. In the experiments using synthetic “unligated” samples, we observed that strong mutant bands could be distinguished visually when the detection fluid did not contain competing oligonucleotides. Conversely, in the case that our detection fluid contained competing oligonucleotides, the signal at the mutant bands was not visible and was comparable to the signal of the negative controls (Fig. 1d). The Python-based software analysis showed concordant results with our visual observations with outputted intensities (n=3, mean ± SE) at the mutant bands at 0.17 ± 0.11 and 16.3 ± 2.7 a.u. for the detection sample tube with and without competing oligonucleotides, respectively. In summary, we have demonstrated a proof-of-concept custom LFT prototype that successfully delivers fluid sequentially and contains fluid within the device. However, we encountered some inconsistent flow across our replicates which will require further 3D printer quality optimization. Regardless, our custom LFT prototype presents competitive cost features compared to other commercial projects. Our current prototype costs $USD0.25 per unit and uses biodegradable plastics with safe plastic pins to release the fluid from the custom tube caps with sealed foils. In contrast, commercial amplicon-contained LFTs cost $USD15 per unit and use metal blades to puncture plastic tubes, requiring thicker high-quality plastic to ensure user safety [3].


Acknowledgements (Optional): : We thank Dr. David McIntyre for his prior contributions to the earlier designs of the custom LFT device; Dr. Annie Wong-On-Wing, Annapurni Sriram, and Dr. Amy Oreskovic for their contributions to assay chemistries; Parker Ruth and Brian Tran for software analysis; Dr. Lisa Frenkel and her group at Seattle Children’s Research Institute for their expertise in OLA for HIVDR. This work was funded by NIH R01 (Lutz: 5R01AI145486). The funder has no role in the study design or result interpretation of this work.

References (Optional): : [1] Beck IA, Mahalanabis M, Pepper G, Wright A, Hamilton S, Langston E, Frenkel LM. Rapid and sensitive oligonucleotide ligation assay for detection of mutations in human immunodeficiency virus type 1 associated with high-level resistance to protease inhibitors. J Clin Microbiol. 2002 Apr;40(4):1413-9. doi: 10.1128/JCM.40.4.1413-1419.2002. PMID: 11923366; PMCID: PMC140364.



[2] Panpradist N, Beck IA, Vrana J, Higa N, McIntyre D, Ruth PS, So I, Kline EC, Kanthula R, Wong-On-Wing A, Lim J, Ko D, Milne R, Rossouw T, Feucht UD, Chung M, Jourdain G, Ngo-Giang-Huong N, Laomanit L, Soria J, Lai J, Klavins ED, Frenkel LM, Lutz BR. OLA-Simple: A software-guided HIV-1 drug resistance test for low-resource laboratories. EBioMedicine. 2019 Dec;50:34-44. doi: 10.1016/j.ebiom.2019.11.002. Epub 2019 Nov 22. PMID: 31767540; PMCID: PMC6921160.



[3] TwistDx. U-Star Disposable Nucleic Acid Lateral Flow Detection Units. Accessed on April 30th, 2022. https://www.twistdx.co.uk/product/u-star-disposable-nucleic-acid-lateral-flow-detection-units-pack-of-20/