Rapid analysis of Escherichia coli O157:H7 using isothermal recombinase polymerase amplification combined with triple-labeled nucleotide probes

Hu, Jinqiang; Wang, Yi; Su, Haijian; Ding, Huimin; Sun, Xiaoyan; Gao, Hui; Geng, Yangyang; Wang, Zhangcun

doi:10.1016/j.mcp.2019.101501

Cited by 28 publications

(25 citation statements)

References 25 publications

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“…A recent, comprehensive review of the RPA technique highlights how RPA has been used to amplify DNA and RNA (by prior reverse transcription) from an array of bacterial, viral and metazoan target sequences, with examples of single cell sensitivity, and a positive result within a few minutes (Li et al 2019). E. coil-specific RPA has so far been limited to the detection of O157:H7 (Choi et al 2016;Hu et al 2020) using target DNA sequences that are not representative of general E. coli populations and, to the best of our knowledge, no such RPA method has been described that could be applied to faecal indicator E. coli testing.…”

Section: Accepted Articlementioning

confidence: 99%

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Recombinase polymerase amplification for fast, selective, DNA‐based detection of faecal indicator Escherichia coli

McQuillan

Wilson

2021

Lett Appl Microbiol

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The bacterium Escherichia coli is commonly associated with the presence of faecal contamination in environmental samples, and is therefore subject to statutory surveillance. This is normally done using a culture‐based methodology, which can be slow and laborious. Nucleic acid amplification for the detection of E. coli DNA sequences is a significantly more rapid approach, suited for applications in the field such as a point of sample analysis, and to provide an early warning of contamination. An existing, high integrity qPCR method to detect the E. coli ybbW gene, which requires almost an hour to detect low quantities of the target, was compared with a novel, isothermal RPA method, targeting the same sequence but achieving the result within a few minutes. The RPA technique demonstrated equivalent inclusivity and selectivity, and was able to detect DNA extracted from 100% of 99 E. coli strains, and exclude 100% of 30 non‐target bacterial species. The limit of detection of the RPA assay was at least 100 target sequence copies. The high speed and simple, isothermal amplification chemistry may indicate that RPA is a more suitable methodology for on‐site E. coli monitoring than an existing qPCR technique.

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Section: Accepted Articlementioning

confidence: 99%

“…2016; Hu et al . 2020) using target DNA sequences that are not representative of general E. coli populations and, to the best of our knowledge, no such RPA method has been described that could be applied to faecal indicator E. coli testing.…”

Section: Introductionmentioning

confidence: 99%

Recombinase polymerase amplification for fast, selective, DNA‐based detection of faecal indicator Escherichia coli

McQuillan

Wilson

2021

Lett Appl Microbiol

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“…Improvements upon the traditional RPA + LFA setup include work by the Wang research group, which applied surface-enhanced Raman scattering (SERS) to an LFA to simultaneously detect L. monocytogenes and Salmonella enteritidis serotype Enteritidis following RPA. 107 SERS enhances the Raman scattering of molecules adsorbed to the structures. Analysis of the Raman scattering through spectroscopy allows for highly sensitive and quantitative analysis of the bacteria.…”

Section: Paper-based Detection Methodsmentioning

confidence: 99%

Point-of-Need Diagnostics for Foodborne Pathogen Screening

et al. 2021

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“…2017; Hu et al . 2020). RPA couples isothermal recombinase‐driven primer targeting the template with strand‐displacement DNA synthesis, which can achieve exponential amplification (Xia et al .…”

Section: Introductionmentioning

confidence: 99%

“…Recently, recombinase polymerase amplification (RPA), as a novel isothermal amplification technology of nucleic acid for the diagnosis of infectious disease in DNA or RNA levels (Law et al 2018;Kissenkotter et al 2020), has drawn great attention due to its high specificity and sensitivity, constant temperature (37-42°C), rapid detection (20-30 min) and popularization (Wang et al 2017;Hu et al 2020). RPA couples isothermal recombinase-driven primer targeting the template with strand-displacement DNA synthesis, which can achieve exponential amplification (Xia et al 2014;Lai and Lau 2020).…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

Zhang

et al. 2021

Letters in Applied Microbiology

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Tuberculosis (TB), as a common infectious disease, still remains a severe challenge to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real‐time recombinase polymerase amplification (RPA) technology for identifying M. tuberculosis within 20 min at 39°C via custom‐designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4‐R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify M. tuberculosis with the detection limit of 4·0 copies per μl, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B‐based RPA could determine the genomic DNA extracted from M. tuberculosis with high reliability (100%, 22/22). More importantly, when testing clinical sputum samples, the real‐time RPA displayed an admirable sensitivity (90%, 95% CI: 80·0‐96·0%) and specificity (98%, 95% CI: 89·0‐100·0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real‐time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.

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Rapid analysis of Escherichia coli O157:H7 using isothermal recombinase polymerase amplification combined with triple-labeled nucleotide probes

Cited by 28 publications

References 25 publications

Recombinase polymerase amplification for fast, selective, DNA‐based detection of faecal indicator Escherichia coli

Recombinase polymerase amplification for fast, selective, DNA‐based detection of faecal indicator Escherichia coli

Point-of-Need Diagnostics for Foodborne Pathogen Screening

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

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