Strategies for highly sensitive biomarker detection by Rolling Circle Amplification of signals from nucleic acid composed sensors

Stougaard, Magnus; Juul, Sissel; Andersen, Félicie F.; Knudsen, Birgitta R.

doi:10.1039/c1ib00049g

Cited by 44 publications

(38 citation statements)

References 205 publications

(522 reference statements)

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“…In recent years alternative isothermal amplification methods which can be categorized into (i) T7 promoter-driven amplifications (transcription-mediated amplification [TMA], nucleic acid sequence-based amplification [NASBA], and single primer isothermal amplification [SPIA]), (ii) strand displacement methods (strand displacement amplification [SDA], loop-mediated isothermal amplification [LAMP], and smart amplification [SmartAmp]), (iii) helicase-dependent amplification (HDA), (iv) recombinase polymerase amplification (RPA), and (v) rolling-circle amplification (RCA) methods (8)(9)(10)(11)(12) have been developed. Some were purposely designed for isothermal amplification starting from RNA (TMA, NASBA, and SPIA), whereas others initially targeted DNA (SDA, LAMP, HDA, RPA, and RCA) and were only later adapted for RNA targets.…”

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confidence: 99%

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

et al. 2013

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Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. Syndromic panels for infectious and emerging infectious diseases have been suggested to be of value in point-of-care (POC) diagnostics for developing countries and for biodefense (1). Since the introduction of molecular diagnostics and in particular real-time PCR, ample proof of its sensitivity and specificity has been generated. Indeed, molecular diagnostics are deemed superior to bacterial culture techniques or serological diagnostics (2-4). It has even been suggested to entirely eliminate the old methods in order to streamline centralized laboratories for molecular diagnostics (5-7).In recent years alternative isothermal amplification methods which can be categorized into (i) T7 promoter-driven amplifications (transcription-mediated amplification [TMA], nucleic acid sequence-based amplification [NASBA], and single primer isothermal amplification [SPIA]), (ii) strand displacement methods (strand displacement amplification [SDA], loop-mediated isothermal amplification [LAMP], and smart amplification [SmartAmp]), (iii) helicase-dependent amplification (HDA), (iv) recombinase polymerase amplification (RPA), and (v) rolling-circle amplification (RCA) methods (8-12) have been developed. Some were purposely designed for isothermal amplification starting from RNA (TMA, NASBA, and SPIA), whereas others initially targeted DNA (SDA, LAMP, HDA, RPA, and RCA) and were only later adapted for RNA targets. Nonspecific intercalating fluorophores or fluorescent primers have been used for real-time detection in LAMP, SDA, HDA, and RCA, and specific detection probe formats have been developed for NASBA, RCA, HDA,.In isothermal and exponential RPA, the phage recombinase UvsX and its cofactor UvsY form a nucleoprotein complex with oligonucleotide primers to scan for homologous sequences in a DNA template. Recognition of a specific homologous sequence leads to the initiation of strand invasion of the com...

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Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

et al. 2013

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“…Various other readout systems for RCA are nicely described in Stougaard et al [160]. Mazutis et al described a droplet-based microfluidic system for single molecule amplification by RCA [161].…”

Section: Lateral Flow Strip Through Dual Immunoreactionsmentioning

confidence: 99%

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

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Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results.However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis.In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids. miniaturized systems can be diverse. The majority of publications are focussed on clinical diagnostics including foodborne organisms for environmental monitoring [10][11][12]. In case of an infectious disease, cultivation and phenotypic characterization are still the standard methods of microbiologists which need days up to weeks to obtain a diagnostic result. Hence, scientists started to make use of nucleic acid amplification methods to accelerate the analysis. The most widespread and well known technique for this purpose is the polymerase chain reaction (PCR) [10,13], which exploits the activity of the DNA polymerase. The method is based on a thermal cycling process consisting of repeated cycles of heating and cooling steps allowing for DNA melting, sequence specific primer binding and enzymatic amplification of the target nucleic acid. The quantitative assessment of target copies is possible by using a real-time PCR approach, where a concentration dependent signal (e.g. fluorescence) increases over time [14]. The latter enables the user to easily assess the pathogen load of patient samples [15][16][17][18]. Since the first example of a PCR on a miniaturized system in 1994 [19,20], numerous devices were presented, which integrate not just the amplification, but also sample preparation and detection steps [9,[21][22][23][24][25][26][27][28]. Just a few of those microsystems have reached the market so far, but some have shown a fascinating potential to replace the classical laboratory-oriented state-of-the art [29][30][31][32][33]. setups has been shown to obtain a similar result [34]. A smaller heat capacity allows for rapid changes in temperature beneficial for the PCR time as well as a higher parallelism of multiple genetic samples [34].However, a major drawback of the integration of PCR to microsystems is the necessity of sophisticated instrumentation. The reaction requires a thermal cycling instrumentation, space and considerable expertise [35]. Therefore, isothermal reactions for the ta...

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“…Altenative several isothermal amplification methods that have been developed in the recent decade include T7 promotor driven amplifcations (TMA, NASBA, SPIA) strand displacement methods (SDA, LAMP, SmartAmp), helicase dependent amplification (HDA), recombinase polymerase amplification (RPA) and several rolling circle amplification (RCA) methods [23][24][25][26][27] .…”

Section: Minaturising Molecular Diagnostic Assaysmentioning

confidence: 99%

Global health

Kappas¹,

Groß²,

Kelleher³

2012

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Strategies for highly sensitive biomarker detection by Rolling Circle Amplification of signals from nucleic acid composed sensors

Cited by 44 publications

References 205 publications

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Global health

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