Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinaseﾠpolymeraseﾠamplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sampleﾠconcentrationﾠusing aﾠstandard curveﾠIn this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest

A Lateral Flow Assay for Quantitative Detection of Amplified HIV-1 RNA

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

NPM1 directs PIDDosome-dependent caspase-2 activation in the nucleolus

The PIDDosome (PIDD–RAIDD–caspase-2 complex) is considered to be the primary signaling platform for caspase-2 activation in response to genotoxic stress. Yet studies of PIDD-deficient mice show that caspase-2 activation can proceed in the absence of PIDD. Here we show that DNA damage induces the assembly of at least two distinct activation platforms for caspase-2: a cytoplasmic platform that is RAIDD dependent but PIDD independent, and a nucleolar platform that requires both PIDD and RAIDD. Furthermore, the nucleolar phosphoprotein nucleophosmin (NPM1) acts as a scaffold for PIDD and is essential for PIDDosome assembly in the nucleolus after DNA damage. Inhibition of NPM1 impairs caspase-2 processing, apoptosis, and caspase-2–dependent inhibition of cell growth, demonstrating that the NPM1-dependent nucleolar PIDDosome is a key initiator of the caspase-2 activation cascade. Thus we have identified the nucleolus as a novel site for caspase-2 activation and function

A paper and plastic device for performing recombinase polymerase amplification of HIV DNA

Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 minutes. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings

Sequences used for lateral flow assay and NASBA.

<p>Sequences used for lateral flow assay and NASBA.</p

Optimized experimental conditions for the lateral flow detection assay.

<p>Optimized experimental conditions for the lateral flow detection assay.</p

Lateral flow assay design.

<p>The lateral flow assay is designed to detect a 142 bp amplified RNA sequence<b>.</b> 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.</p

Performance of the optimized lateral flow assay.

<p>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 <i>in vitro</i> 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.</p

Paper-based detection of HIV-1 drug resistance using isothermal amplification and an oligonucleotide ligation assay

Regular HIV-1 viral load monitoring is the standard of care to assess antiretroviral therapy effectiveness in resource-rich settings. Persistently elevated viral loads indicate virologic failure (VF), which warrants HIV drug resistance testing (HIVDRT) to allow individualized regimen switches. However, in settings lacking access to HIVDRT, clinical decisions are often made based on symptoms, leading to unnecessary therapy switches and increased costs of care. This work presents a proof-of-concept assay to detect M184V, the most common drug resistance mutation after first-line antiretroviral therapy failure, in a paper format. The first step isothermally amplifies a section of HIV-1ﾠreverse transcriptaseﾠcontaining M184V using a recombinase polymerase amplification (RPA) assay. Then, an oligonucleotide ligation assay (OLA) is used to selectively label the mutant and wild type amplified sequences. Finally, a lateral flow enzyme-linked immunosorbent assay (ELISA) differentiates between OLA-labeled products with or without M184V. Our method shows 100% specificity and 100% sensitivity when tested with samples that contained 200 copies of mutant DNA and 800 copies of wild type DNA prior to amplification. When integrated with sample preparation, this method may detect HIV-1 drug resistance at a low cost and at a rural hospital laboratory