Colorimetric detection of both total genomic and loci-specific DNA methylation from limited DNA inputs

Wee, Eugene J. H.; Ngo, Thu Ha; Trau, Matt

doi:10.1186/s13148-015-0100-6

Cited by 43 publications

(48 citation statements)

References 35 publications

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“…RPA can be used to amplify double stranded DNA, single stranded DNA, methylated DNA [16], cDNA generated through reverse transcription of RNA or miRNA [17] (Tables 2e6). There are several reverse transcriptases that have been used with RPA, including Transcriptor R (Roche), Sensiscript R (Qiagen), or MuLV R (Applied Biosystems), with initial reports demonstrating that Transcriptor provides the best performance. cDNA can be produced prior to RPA or in the same reaction [18,19] and RT-Freeze is also available from TwistDx.…”

Section: Sample Typesmentioning

confidence: 99%

Recombinase polymerase amplification: Basics, applications and recent advances

Lobato

O’Sullivan

2018

TrAC Trends in Analytical Chemistry

734

522

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a b s t r a c tRecombinase polymerase amplification (RPA) is a highly sensitive and selective isothermal amplification technique, operating at 37e42 C, with minimal sample preparation and capable of amplifying as low as 1e10 DNA target copies in less than 20 min. It has been used to amplify diverse targets, including RNA, miRNA, ssDNA and dsDNA from a wide variety of organisms and samples. An ever increasing number of publications detailing the use of RPA are appearing and amplification has been carried out in solution phase, solid phase as well as in a bridge amplification format. Furthermore, RPA has been successfully integrated with different detection strategies, from end-point lateral flow strips to real-time fluorescent detection amongst others. This review focuses on the different methodologies and advances related to RPA technology, as well as highlighting some of the advantages and drawbacks of the technique.

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Section: Sample Typesmentioning

confidence: 99%

Recombinase polymerase amplification: Basics, applications and recent advances

Lobato

O’Sullivan

2018

TrAC Trends in Analytical Chemistry

734

522

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“…However, to avoid the instrumentational limitations of PCR, isothermal approaches such as the recombinase polymerase amplification (RPA)7 which uses enzymes instead of heat to achieve exponential amplification have been developed. With PCR-like sensitivity, yet requiring only a low constant working temperature, RPA is potentially useful for point-of-care (POC) applications89101112. However, there is still a need to develop simpler RPA assays for plant pathogen detection in the field comprising all steps, from DNA isolation to visualization of results.…”

mentioning

confidence: 99%

Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes

Lau

Wee

et al. 2017

Sci Rep

Self Cite

143

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Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

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“…55,57,102,123,[206][207][208] RPA, unlike PCR, relies on enzymes, at a single low temperature, to separate dsDNA, assist in primer/target recognition and primer extension. 45 The advantages of RPA include highly efficient amplification and low constant operating temperature, 45 thus making it a candidate for POC applications.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, RPA is a highly sensitive with a detection limit as low as 6.25 fg of genomic DNA input with specificity >95%. 101 In addition, POC-compatible readouts such as portable fluorometers and equipment-free naked eye strategies 55,123 have also been used with RPA to enable field applications.…”

Section: Introductionmentioning

confidence: 99%

“…17,18 As an alternative to PCR, recombinase polymerase amplification (RPA) using DNA repair enzymes to replace high temperature heating in DNA denaturation 45 achieves rapid DNA amplification at low constant temperature (37°C -42°C) and PCR-like sensitivity, making it uniquely suited for point-of-care (POC) applications. While it is evident that RPA has high potential in POC diagnostics, 57,[123][124][125] there is still a need to develop simpler rapid DNA-based diagnostic assay for plant pathogen detection in the field comprising all steps, from DNA isolation to visualization of the amplification results.…”

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confidence: 99%

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Development of point-of-care and multiplex diagnostic methods for the detection of plant pathogens

Lau¹

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New diagnostic technologies for the detection of plant pathogens with high multiplexing ability and point-of-care (POC) capability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and highthroughput multiplex detection capability. The aim of this project is to develop new methods combining nucleic acid amplification, multiplexing and point-of-care application for the detection of plant pathogens.Isothermal amplification methods, such as recombinase polymerase amplification (RPA) which operates at low constant temperature are especially suited for POC applications. However, the development of a rapid and simplified detection method for the isothermal amplification products in resource-poor settings is still challenging. A new method to visualize the success of the amplification step, and therefore the presence of pathogen DNA in a sample, was developed based on bridging flocculation. This method requires minimal equipment and can be assessed by the naked eye allowing its use in POC settings. The method was initially applied to the rapid and sensitive detection of several important plant pathogens and subsequently extended to animal and human pathogens to demonstrate the wide applications of the approach. A nanoparticle based electrochemical biosensor was also developed for rapid and sensitive detection of amplified plant pathogen DNA on screen-printed carbon electrodes. Gold nanoparticles were employed as a probe for electrochemical assessment by differential pulse voltammetry (DPV). This method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible.To allow multiplex detection of plant pathogen, a novel method that can screen for thousands of plant pathogens with high specificity and sensitivity using molecular inversion probes (MIPs) was been developed. As proof of concept, a MIP targeting a unique DNA sequence present in the F.oxysporum f.sp. conglutinans (Foc) genome was designed. The specificity, sensitivity and detection limit of the assay were assessed and used to detect the presence of pathogen in infected Arabidopsis thaliana samples. This methodology successfully detected as little as 2.5 ng of pathogen DNA and it was highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. Finally, a diagnostic platform for POC plant pathogen detection using a combination of surface-enhanced Raman ii scattering (SERS) and RPA was developed for rapid multiplex detection. The RPA-SERS method was faster, more sensitive than polymerase chain reaction and could detect as little as 2 copies of...

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Colorimetric detection of both total genomic and loci-specific DNA methylation from limited DNA inputs

Cited by 43 publications

References 35 publications

Recombinase polymerase amplification: Basics, applications and recent advances

Recombinase polymerase amplification: Basics, applications and recent advances

Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes

Development of point-of-care and multiplex diagnostic methods for the detection of plant pathogens

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