Here we used a SlipChip microfluidic device to evaluate the performance of digital reverse transcription-loop mediated isothermal amplification (dRT-LAMP) for quantification of HIV viral RNA. Tests are needed for monitoring HIV viral load to control the emergence of drug resistance and to diagnose acute HIV infections. In resource-limited settings, in vitro measurement of HIV viral load in a simple format is especially needed, and single-molecule counting using a digital format could provide a potential solution. We showed here that when one-step dRT-LAMP is used for quantification of HIV RNA, the digital count is lower than expected and is limited by the yield of desired cDNA. We were able to overcome the limitations by developing a microfluidic protocol to manipulate many single molecules in parallel through a two-step digital process. In the first step we compartmentalize the individual RNA molecules (based on Poisson statistics) and perform reverse transcription on each RNA molecule independently to produce DNA. In the second step, we perform the LAMP amplification on all individual DNA molecules in parallel. Using this new protocol, we increased the absolute efficiency (the ratio between the concentration calculated from the actual count and the expected concentration) of dRT-LAMP 10-fold, from ~2 % to ~23 %, by i) using a more efficient reverse transcriptase, ii) introducing RNase H to break up the DNA:RNA hybrid, and iii) adding only the BIP primer during the RT step. We also used this two-step method to quantify HIV RNA purified from 4 patient samples and found that in some cases, the quantification results were highly sensitive to the sequence of the patient’s HIV RNA. We learned the following three lessons from this work: i) digital amplification technologies, including dLAMP and dPCR, may give adequate dilution curves and yet have low efficiency, thereby providing quantification values that underestimate the true concentration. Careful validation is essential before a method is considered to provide absolute quantification; ii) the sensitivity of dLAMP to the sequence of the target nucleic acid necessitates additional validation with patient samples carrying the full spectrum of mutations; and iii) for multistep digital amplification chemistries, such as a combination of reverse transcription with amplification, microfluidic devices may be used to decouple these steps from one another and to perform them under different, individually optimized conditions for improved efficiency.
There is a need for new quantitative in vitro models of drug uptake and diffusion to help assess drug toxicity/efficacy as well as new more predictive models for drug discovery. We report a three-dimensional (3D) multilayer spheroid model and a new algorithm to quantitatively study uptake and inward diffusion of fluorescent calcein via gap junction intercellular communication (GJIC). When incubated with calcein-AM, a substrate of the efflux transporter P-glycoprotein (Pgp), spheroids from a variety of cell types accumulated calcein over time. Accumulation decreased in spheroids overexpressing Pgp (HEK-MDR) and was increased in the presence of Pgp inhibitors (verapamil, loperamide, cyclosporin A). Inward diffusion of calcein was negligible in spheroids that lacked GJIC (OVCAR-3, SK-OV-3) and was reduced in the presence of an inhibitor of GJIC (carbenoxolone). In addition to inhibiting Pgp, verapamil and loperamide, but not cyclosporin A, inhibited inward diffusion of calcein, suggesting that they also inhibit GJIC. The dose response curves of verapamil’s inhibition of Pgp and GJIC were similar (IC50: 8 μM). The method is amenable to many different cell types and may serve as a quantitative 3D model that more accurately replicates in vivo barriers to drug uptake and diffusion.
Diagnostic assays are an important part of health care, both in the clinic and in research laboratories. In addition to improving treatments and clinical outcomes, rapid and reliable diagnostics help track disease epidemiology, curb infectious outbreaks, and further the understanding of chronic illness. Disease markers such as antigens, RNA, and DNA are present at low concentrations in biological samples, such that the majority of diagnostic assays rely on an amplification reaction before detection is possible. Ideally, these amplification reactions would be sensitive, specific, inexpensive, rapid, integrated, and automated. Microfluidic technology currently in development offers many advantages over conventional benchtop reactions that help achieve these goals. The small reaction volumes and energy consumption make reactions cheaper and more efficient in a microfluidic reactor. Additionally, the channel architecture could be designed to perform multiple tests or experimental steps on one integrated, automated platform. This review explores the current research on microfluidic reactors designed to aid diagnostic applications, covering a broad spectrum of amplification techniques and designs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.