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Rosser A.,London School of Hygiene and Tropical Medicine | Forrest M.,TwistDX Ltd
Parasites and Vectors | Year: 2015

Background: Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs. Amplification of schistosome DNA in urine by PCR is sensitive and specific but requires infrastructure, financial resources and skilled personnel, often not available in endemic areas. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is simple, rapid, portable and needs few resources. Findings: Here a Schistosoma haematobium RPA assay was developed and adapted so that DNA amplicons could be detected using oligochromatographic Lateral Flow (LF) strips. The assay successfully amplified S. haematobium DNA at 30-45 °C in 10 mins and was sensitive to a lower limit of 100 fg of DNA. The assay was also successful with the addition of crude urine, up to 5 % of the total reaction volume. Cross amplification occurred with other schistosome species but not with other common urine microorganisms. Conclusion: The LF-RPA assay developed here can amplify and detect low levels of S. haematobium DNA. Reactions are rapid, require low temperatures and positive reactions are interpreted using lateral flow strips, reducing the need for infrastructure and resources. This together with an ability to withstand inhibitors within urine makes RPA a promising technology for further development as a molecular diagnostic tool for urogenital schistosomiasis. © 2015 Rosser et al.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-FP-SICA | Phase: HEALTH.2011.2.3.3-2 | Award Amount: 7.35M | Year: 2011

Current surveillance systems and control efforts are clearly insufficient to combat dengue in endemic countries and to prevent spread to previously uninfected areas (including Europe). We will focus on the following aspects of the call: better diagnosis, surveillance, prevention as well as prediction and/or prevention of the spread of Dengue fever to previously uninfected regions (including Europe) in the context of climate change. We will develop a rapid diagnostic assay for resource limited settings that can be used as point-of-care, is portable, provides rapid results and is inexpensive. Novel tools for vector monitoring will be tested. We propose to develop a comprehensive, early warning, laboratory-based sentinel surveillance system that integrates clinical, entomological, environmental, socio-economic, and weather/climate indices to evaluate predictive capability for epidemic dengue. The aim is to translate this information directly into improved tools for surveillance (early diagnostic assays, early warning systems and predictive models, risk maps, improved tools for vector surveillance). Furthermore, we will test the impact of a novel community-based strategy on the reduction of dengue incidence in school aged children. Lastly, the integrated information on trends of importation of dengue, global mobility patterns, and vector distribution under changing climate conditions will improve our current understanding of the risk of introduction into previously uninfected areas, in particular Europe. Web based predictive tools, models and maps will be produced. The information will be disseminated to relevant agencies, academia and policy makers and will translate into improved surveillance and control.


Shen F.,University of Chicago | Davydova E.K.,University of Chicago | Du W.,University of Chicago | Kreutz J.E.,University of Chicago | And 2 more authors.
Analytical Chemistry | Year: 2011

In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for "yes or no" digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37-42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader range of applications. © 2011 American Chemical Society.


Patent
TwistDx Inc. | Date: 2010-04-30

This disclosure describe three related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of the bacterial RecA and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods has the advantage of not requiring thermocycling or thermophilic enzymes. Further, the improved processivity of the disclosed methods allow amplification of DNA up to hundreds of megabases in length.


Trademark
TwistDX Ltd | Date: 2015-06-05

Reagents and enzymes for nucleic acid amplification and detection, for scientific, research and industrial use. Reagents and enzymes for nucleic acid amplification and detection, for medical and diagnostic use. Nucleic acid amplification apparatus for scientific research use.


Trademark
TwistDx Ltd | Date: 2013-06-21

Nucleic acid amplification apparatus for scientific research use. Nucleic acid amplification apparatus for medical diagnostic use.


Trademark
TwistDx Ltd | Date: 2013-05-21

Reagents and enzymes for nucleic acid amplification and detection, for scientific, research and industrial use. Reagents and enzymes for nucleic acid amplification and detection, for medical diagnostic use.


Trademark
TwistDx Ltd | Date: 2013-05-21

Reagents and enzymes for nucleic acid amplification and detection, for scientific, research and industrial use. Reagents and enzymes for nucleic acid amplification and detection, for medical and diagnostic use.


Trademark
TwistDx Ltd | Date: 2013-05-21

Reagents and enzymes for nucleic acid amplification and detection, for scientific, research and industrial use. Reagents and enzymes for nucleic acid amplification and detection, for medical diagnostic use.


Patent
TwistDx Inc. | Date: 2010-06-21

This disclosure provides novel reversibly terminated ribonucleotides which can be used as a reagent for DNA sequencing reactions. Methods of sequencing nucleic acids using the disclosed nucleotides are also provided.

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