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

Crannell, Zachary; Rohrman, Brittany A.; Richards‐Kortum, Rebecca

doi:10.1021/ac5011298

Cited by 86 publications

(80 citation statements)

References 13 publications

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“…Several proof-of-concept studies have been performed to perform isothermal reactions in paper, incubate the reaction using body heat, or detect amplified HIV DNA on lateral flow strips. 331–337 However, the complexities of biological samples and the need for high sensitivity again limit the current usefulness of these technologies. In a thorough review by Craw & Balachandran, 330 the authors conclude that although several isothermal nucleic acid amplification techniques have been extensively validated, the limiting factor is the integration of upstream sample preparation and nucleic acid extraction with downstream detection techniques.…”

Section: Diagnostics For Neonatal Healthmentioning

confidence: 99%

Point-of-care diagnostics to improve maternal and neonatal health in low-resource settings

et al. 2017

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Each day, approximately 830 women and 7,400 newborns die from complications during pregnancy and childbirth. Improving maternal and neonatal health will require bringing rapid diagnosis and treatment to the point of care in low-resource settings. However, to date there are few diagnostic tools available that can be used at the point of care to detect the leading causes of maternal and neonatal mortality in low-resource settings. Here we review both commercially available diagnostics and technologies that are currently in development to detect the leading causes of maternal and neonatal mortality, highlighting key gaps in development where innovative design could increase access to technology and enable rapid diagnosis at the bedside.

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Section: Diagnostics For Neonatal Healthmentioning

confidence: 99%

Point-of-care diagnostics to improve maternal and neonatal health in low-resource settings

et al. 2017

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“…Isothermal methods can amplify nucleic acids at a constant (and relatively lower, if compared with qPCR) temperature using a simple water bath or heat block: in the case of RPA, successful amplification using simply the body heat of the operator has been proven possible (Crannell et al 2014). A wide range of approaches are available for the qualitative detection of LAMP and RPA products, including gel electrophoresis, in-tube fluorescence, lateral flow detection with immunochromatographic strips (Craw and Balachandran, 2012) and also by naked eye using pH-sensitive dyes (Tanner et al 2015).…”

Section: Cost Of Reagents and Equipment And The Issue Of Target Quantmentioning

confidence: 99%

Focusing nucleic acid-based molecular diagnostics and xenomonitoring approaches for human helminthiases amenable to preventive chemotherapy

et al. 2016

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The current mainstay for control of the four major helminth diseases in humans (lymphatic filariasis, onchocerciasis, soiltransmitted helminthiases and schistosomiasis) is with preventive chemotherapy by mass administration of key anthelminthics. Following the London Declaration on Neglected Tropical Diseases in 2012, a roadmap for the elimination and control of these helminthiases by 2020 has been devised. With expected declines in prevalence and intensity of these infections, there is urgent need for implementing more sensitive, high-throughput and cost-effective diagnostic tools. Currently available diagnostic approaches for surveying, monitoring and evaluating helminth control programmes are based on microscopical observation of eggs/larvae, and/or detection of antibodies or parasite antigens in stool, urine or blood; all relatively low-throughput and of limited sensitivity and specificity. Newly proposed approaches for helminthiases diagnosis include the nucleic acid-based methods of (multiplex) real-time polymerase chain reaction assays, loop-mediated isothermal amplification and recombinase polymerase amplification. However, as well as sensitivity/specificity evaluation, their comparison to current 'gold standard' diagnostics and future application in individual-/community-based diagnosis, or in xenomonitoring requires consideration of relative costs, agreement of standard methods and strategic interpretation of resulting data before control/elimination programmes might best utilize molecular diagnostics to inform decision making. We review current nucleic-acid-based molecular diagnostic methods and highlight the needs and future research required to refine these tools for monitoring and evaluation of control and elimination programmes for four major human helminthiases.

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“…NOTE: For this assay, Cryptosporidium parvum, an intestinal parasite, was chosen to serve as the template for generation of the IPC sequence because this organism is unlikely to be found in blood and was available in the lab from another project. To generate the IPC sequence, extract and purify DNA from C. parvum oocysts as described elsewhere 18 . 2.…”

Section: Develop An Internal Positive Controlmentioning

confidence: 99%

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

Crannell

Rohrman

Richards‐Kortum

2015

JoVE

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It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In 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. Video LinkThe video component of this article can be found at

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

Cited by 86 publications

References 13 publications

Point-of-care diagnostics to improve maternal and neonatal health in low-resource settings

Point-of-care diagnostics to improve maternal and neonatal health in low-resource settings

Focusing nucleic acid-based molecular diagnostics and xenomonitoring approaches for human helminthiases amenable to preventive chemotherapy

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

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