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

T. gondii RP Promoters & Knockdown Reveal Molecular Pathways Associated with Proliferation and Cell-Cycle Arrest

Molecular pathways regulating rapid proliferation and persistence are fundamental for pathogens but are not elucidated fully in Toxoplasma gondii. Promoters of T. gondii ribosomal proteins (RPs) were analyzed by EMSAs and ChIP. One RP promoter domain, known to bind an Apetela 2, bound to nuclear extract proteins. Promoter domains appeared to associate with histone acetyl transferases. To study effects of a RP gene's regulation in T. gondii, mutant parasites (Δrps13) were engineered with integration of tetracycline repressor (TetR) response elements in a critical location in the rps13 promoter and transfection of a yellow fluorescent-tetracycline repressor (YFP-TetR). This permitted conditional knockdown of rps13 expression in a tightly regulated manner. Δrps13 parasites were studied in the presence (+ATc) or absence of anhydrotetracycline (-ATc) in culture. -ATc, transcription of the rps13 gene and expression of RPS13 protein were markedly diminished, with concomitant cessation of parasite replication. Study of Δrps13 expressing Myc-tagged RPL22, -ATc, showed RPL22 diminished but at a slower rate. Quantitation of RNA showed diminution of 18S RNA. Depletion of RPS13 caused arrest of parasites in the G1 cell cycle phase, thereby stopping parasite proliferation. Transcriptional differences ±ATc implicate molecules likely to function in regulation of these processes. In vitro, -ATc, Δrps13 persists for months and the proliferation phenotype can be rescued with ATc. In vivo, however, Δrps13 could only be rescued when ATc was given simultaneously and not at any time after 1 week, even when L-NAME and ATc were administered. Immunization with Δrps13 parasites protects mice completely against subsequent challenge with wildtype clonal Type 1 parasites, and robustly protects mice against wildtype clonal Type 2 parasites. Our results demonstrate that G1 arrest by ribosomal protein depletion is associated with persistence of T. gondii in a model system in vitro and immunization with Δrps13 protects mice against subsequent challenge with wildtype parasites

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

Isothermal Nucleic Acid Assays Based on Nucleic Acid Sequence Based Amplification (NASBA) and Recombinase Polymerase Amplification (RPA) for HIV-1 Diagnosis and Management in Low Resource Settings

Over two-thirds of the estimated 35 million people worldwide infected with HIV live in the developing world. Nucleic acid tests (NATs) are necessary for early infant diagnosis and for monitoring patients receiving therapy. However, NATs cost $50-100 USD per test and require expensive thermal cycling equipment that may be unavailable in the developing world. This thesis presents two low-cost NATs for HIV-1 diagnosis and management that are based on isothermal amplification, which eliminates the need for expensive thermal cycling equipment. In one assay, HIV-1 viral RNA is detected using nucleic acid sequence based amplification (NASBA) and a custom lateral flow test. This assay costs about \$16 USD and only requires a heat block. When coupled with NASBA, the lateral flow test detected concentrations of synthetic RNA spanning the entire clinical range. When the assay was evaluated using pediatric plasma samples, the sensitivity (61%) and limit-of-detection (10,000 HIV-1 copies/mL plasma) were lower because of the genetic diversity of the samples, and the specificity was lower (88%) due to amplicon contamination. In the other assay, HIV-1 proviral DNA is amplified using recombinase polymerase amplification (RPA). This assay, which costs about \$5 per test, was integrated into a paper and plastic microfluidic device. The device was capable of amplifying 10 copies of plasmid HIV-1 DNA to detectable levels in 15 minutes. The assay was then adapted for real-time quantification. On average, the assay predicted sample concentrations within one order of magnitude of the correct concentration. In addition, a method for incubating RPA reactions without external equipment was developed. Using human body heat for incubation, all RPA reactions with 10 copies of plasmid HIV-1 DNA and 95% of reactions with 100 copies of plasmid HIV-1 DNA tested positive. Finally, concentrations of background DNA found in whole blood were shown to prevent the amplification of target DNA by RPA. To address this problem, three sequence-specific capture methods were developed to enrich target DNA concentration relative to background DNA concentration. These methods may be enable detection of high proviral loads in 0.1 mL infant blood samples but require improvement to detect lower proviral loads

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

Quantification of HIV-1 DNA using Real-Time Recombinase Polymerase Amplification

Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been applied for has not yet been implemented to quantify sample concentration using a standard curve. Here we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes the real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within one order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that qRPA may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems

Equipment-Free Incubation of Recombinase Polymerase Amplification Reactions Using Body Heat

The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. In this study, we demonstrated that human body heat may be harnessed to incubate recombinase polymerase amplification (RPA) reactions for isothermal amplification of HIV-1 DNA. After measuring the temperature of mock reactions at 4 body locations, the axilla was chosen as the ideal site for comfortable, convenient incubation. Using commonly available materials, 3 methods for securing RPA reactions to the body were characterized. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block. At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. In a cold room with an ambient temperature of 10 degrees Celsius, all reactions containing 10 copies or 100 copies of HIV-1 DNA tested positive when incubated with body heat. These results suggest that human body heat may provide an extremely low-cost solution for incubating RPA reactions in low resource settings

Performance of body heat versus a heat block for incubating RPA reactions at room temperature.

<p>Performance of body heat versus a heat block for incubating RPA reactions at room temperature.</p