Recombinase polymerase amplification assay for rapid detection of Monkeypox virus

Davi, Saskia Dede; Kissenkötter, Jonas; Faye, Martin; Böhlken-Fascher, Susanne; Stahl–Hennig∥, Christiane; Faye, Oumar; Sall, Amadou Alpha; Weidmann, Manfred; Ademowo, Olusegun G.; Hufert, Frank T.; Czerny, Claus-Peter; Wahed, Ahmed Abd El

doi:10.1016/j.diagmicrobio.2019.03.015

Cited by 106 publications

(97 citation statements)

References 26 publications

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“…As of PCR, the results of RPA amplification can be monitored by real-time fluorescence [75]. Fluorophore Dyes, such as SYBR Green and Eva Green can be employed for real time detection [76,77].…”

Section: Real-time Fluorescence Rpamentioning

confidence: 99%

“…However, these dyes cannot distinguish between amplicons and primer dimers, which can lead to false positive results. Therefore, specific probes are preferred to be used in the RPA reaction, including Exo probe and Fpg probe, named after the enzymes introduced [75,78]. The Exo probe carries a fluorescence group and a fluorescence quenching group, which are respectively combined with a thymine, separated by a tetrahydrofuran (THF) base [79].…”

Section: Real-time Fluorescence Rpamentioning

confidence: 99%

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Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens

You¹,

Li²,

Wang³

et al. 2022

Preprint

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With the advent of molecular technology, several isothermal techniques for fast detecting zoonotic pathogens have been developed. Among them, Recombinase Polymerase Amplification (RPA) is becoming an important technology for rapid, sensitive and economical detection of zoonotic pathogens. RPA technology has the advantage of being implemented in a field-based scenario because the method requires minimal sample preparation and is performed at a constant low temperature (37-42°C). It is rapidly becoming a promising tool for rapid detection and further prevention and control of zoonotic diseases. This article will discuss the principles of RPA technology and its derivatives, including RPA coupled with lateral flow test (RPA-LF), real-time fluorescence RPA, Electrochemical RPA, Flocculation RPA and other technologies, and their applications in detection of zoonotic pathogens for a brief review.

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Section: Real-time Fluorescence Rpamentioning

confidence: 99%

Section: Real-time Fluorescence Rpamentioning

confidence: 99%

Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens

You¹,

Li²,

Wang³

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…It can use several samples, such as blood, serum, plasma, feces, urine. Also, as it is reagents have been freeze dried, the RPA kit can be kept at room temperature for several months (81).…”

Section: Isothermal Amplification Of the Targetmentioning

confidence: 99%

Trends in MERS-CoV, SARS-CoV, and SARS-CoV-2 (COVID-19) Diagnosis Strategies: A Patent Review

Nascimento

Santos

Oliveira

et al. 2020

Front. Public Health

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The emergence of a new coronavirus (SARS-CoV-2) outbreak represents a challenge for the diagnostic laboratories responsible for developing test kits to identify those infected with SARS-CoV-2. Methods with rapid and accurate detection are essential to control the sources of infection, to prevent the spread of the disease and to assist decision-making by public health managers. Currently, there is a wide variety of tests available with different detection methodologies, levels of specificity and sensitivity, detection time, and with an extensive range of prices. This review therefore aimed to conduct a patent search in relation to tests for the detection of SARS-CoV, MERS-CoV, and SARS-CoV-2. The greatest number of patents identified in the search were registered between 2003 and 2011, being mainly deposited by China, the Republic of Korea, and the United States. Most of the patents used the existing RT-PCR, ELISA, and isothermal amplification methods to develop simple, sensitive, precise, easy to use, low-cost tests that reduced false-negative or false-positive results. The findings of this patent search show that an increasing number of materials and diagnostic tests for the coronavirus are being produced to identify infected individuals and combat the growth of the current pandemic; however, there is still a question in relation to the reliability of the results of these tests.

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“…Due to its availability as a ready to use lyophilized enzyme mix, simple incubation requirements as well as the ability to detect target sequences from crude lysates [10], RPA is an ideal candidate for the development of small and inexpensive nucleic acid detection equipment for use at the point of care. Up until now, a multitude of RPA based detection assays targeting bacteria [11][12][13][14], viruses [15][16][17][18][19][20], parasites [21][22][23], and microRNAs [24] have been developed. With the addition of a second primer pair and a corresponding fluorescent probe, RPA assays are also easily duplexed in a single reaction vessel [12,13].…”

Section: Introductionmentioning

confidence: 99%

3D Printed Monolithic Microreactors for Real-Time Detection of Klebsiella pneumoniae and the Resistance Gene blaNDM-1 by Recombinase Polymerase Amplification

et al. 2020

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We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-β-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.

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Recombinase polymerase amplification assay for rapid detection of Monkeypox virus

Cited by 106 publications

References 26 publications

Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens

Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens

Trends in MERS-CoV, SARS-CoV, and SARS-CoV-2 (COVID-19) Diagnosis Strategies: A Patent Review

3D Printed Monolithic Microreactors for Real-Time Detection of Klebsiella pneumoniae and the Resistance Gene blaNDM-1 by Recombinase Polymerase Amplification

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