Development of a Novel One-Tube Isothermal Reverse Transcription Thermophilic Helicase-Dependent Amplification Platform for Rapid RNA Detection

Goldmeyer, James; Kong, Huimin; Tang, Wen

doi:10.2353/jmoldx.2007.070012

Cited by 75 publications

(49 citation statements)

References 12 publications

(16 reference statements)

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“…Nucleic acid amplification through HDA is very robust, and the platform can be applied to both DNA and RNA pathogen amplification. 18,19 We report here the development of the IsoAmp CDAD test using HDA coupled with a simple disposable vertical-flow device, which is designed for instrument-free, cross-contamination-proof detection of amplicons, for rapid identification of toxigenic C. difficile in feces. This assay is based on the amplification of a conserved 5Ј-end fragment of C. difficile tcdA gene.…”

mentioning

confidence: 99%

Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile

Chow¹,

McCloskey

Tong³

et al. 2008

The Journal of Molecular Diagnostics

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103

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Enzyme immunoassays (EIAs) are commonly used for the diagnosis of cases of Clostridium difficile-associated diarrhea (CDAD). However, these EIAs have high falsenegative rates, even in patients with severe clinical disease. We have developed an IsoAmp CDAD test using a simple and user-friendly procedure to identify toxigenic C. difficile in feces. After DNA extraction from fecal samples, both the conserved sequence of the 5-end fragment of the C. difficile tcdA toxin gene and competitive amplification internal control sequence were amplified using helicase-dependent amplification. Amplification products were detected using a novel amplicon-containment detection device. The analytical sensitivity of the assay was 20 copies of C. difficile genomic DNA per reaction. Evaluation of the clinical sensitivity and specificity of the IsoAmp CDAD test versus an EIA method using a PCR method as the reference standard revealed 100% sensitivity and 100% specificity for the IsoAmp CDAD test compared with 90.9% sensitivity and 100% specificity for the EIA method. Because the IsoAmp CDAD test requires no expensive equipments for nucleic acid amplification or detection and can be performed on a random access basis, the test provides a practical alternative to immunoassays for the diagnosis of CDAD with improved sensitivity. (J Mol

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mentioning

confidence: 99%

Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile

Chow¹,

McCloskey

Tong³

et al. 2008

The Journal of Molecular Diagnostics

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103

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“…We saw the appropriate threshold value increases in our quantitative PCR, but not in our HDA. We accepted these results as real based on literature reports that stated that HDA is not always linear in this respect (Chow et al 2008;Goldmeyer et al 2007Goldmeyer et al , 2008Tong et al 2008). These explanations were not appropriate for the observed discrepancies in our data.…”

Section: Discussionmentioning

confidence: 97%

Erratum to: An integrated disposable device for DNA extraction and helicase dependent amplification

Mahalanabis

ALMuayad

et al. 2011

Biomed Microdevices

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In the original manuscript, we reported the demonstration of an integrated microfluidic chip that performed helicase dependent amplification (HDA) on samples containing live bacteria. Bacterial lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter were incorporated into a disposable polymer cartridge format. We reported that the device was able to detect as few as 10 colony-forming units (CFU) of E. coli in growth medium. While the main conclusions of the original paper remain sound, the data presented in support of those conclusions contained errors that we detail, discuss and correct here. In short, we misidentified a non-specific product as a specific product of our HDA reaction. We incorrectly called reactions containing the non-specific product (length 70 bp) positive. Further investigation demonstrated that our primer set was faulty and not capable of amplifying the specific product.Here we redesigned primers, sequenced all of the products and reran all of the experiments reported previously to generate a new, verified dataset.

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“…A sensitivity of as few as 10 copies of bacterial genomic DNA has been presented [103]. This thermophilic HDA system was further developed for diagnostic applications by Goldmeyer et al [106,107]. They reported that the HDA is suitable for the rapid …”

Section: Hda: Helicase-dependent Amplificationmentioning

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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Development of a Novel One-Tube Isothermal Reverse Transcription Thermophilic Helicase-Dependent Amplification Platform for Rapid RNA Detection

Cited by 75 publications

References 12 publications

Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile

Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile

Erratum to: An integrated disposable device for DNA extraction and helicase dependent amplification

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

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