One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

Xie, Jingyu; Yang, Xiaohan; Duan, Lei; Chen, Keyi; Liu, Pan; Zhan, Wenli; Zhang, Changbin; Zhao, Hongyu; Wei, Mengru; Tang, Yi; Luo, Mingyong

doi:10.3389/fmicb.2021.629533

Cited by 5 publications

(1 citation statement)

References 30 publications

(24 reference statements)

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“…The use of lateral flow strips (LFS) as endpoint visual readouts of the amplified DNA targets makes the method less device-dependent. The colored signal can be observed semi-quantitatively by the naked eye on the LFS using gold nanoparticles (AuNPs) that interact with the labeled isothermal amplification product ( Wang et al., 2019 ; Xie et al., 2021 ). RPA-LFS has been successfully used for the molecular diagnosis of diseases caused by pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus , Mycobacterium tuberculosis and neo-cryptococcosis ( Yang et al., 2013 ; Law et al., 2018 ; Ma et al., 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip

Wang

Chen

et al. 2021

Front. Cell. Infect. Microbiol.

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The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site visualization detection method for CPE using improved recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) method, based on four most popular carbapenemase genes: blaKPC, blaNDM, blaOXA-48-like, and blaIMP. All available allelic variants of the above carbapenemases were downloaded from the β-lactamase database, and the conserved regions were used as targets for RPA assay. Five primer sets were designed targeting to each carbapenemase gene and the RPA amplification products were analyzed by agarose gel electrophoresis. FITC-labeled specific probes were selected, combined with the best performance primer set (Biotin-labeled on the reverse primer), and detected by RPA-LFS. Mismatches were made to exclude the false positive signals interference. This assay was evaluated in 207 clinically validated carbapenem-resistant Enterobacterales (CRE) isolates and made a comparison with conventional PCR. Results showed that the established RPA-LFS assay for CPE could be realized within 30 min at a constant temperature of 37°C and visually detected amplification products without the need for special equipment. This assay could specifically differentiate the four classes of carbapenemases without cross-reactivity and shared a minimum detection limit of 100 fg/reaction (for blaKPC, blaNDM, and blaOXA-48-like) or 1000 fg/reaction (for blaIMP), which is ten times more sensitive than PCR. Furthermore, the detection of 207 pre-validated clinically CRE strains using the RPA-LFS method resulted in 134 blaKPC, 69 blaNDM, 3 blaOXA-48-like, and 1 blaIMP. The results of the RPA-LFS assay were in consistent with PCR, indicating that this method shared high sensitivity and specificity. Therefore, the RPA-LFS method for CPE may be a simple, specific, and sensitive method for the rapid diagnosis of carbapenemase Enterobacterales.

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Section: Introductionmentioning

confidence: 99%

Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip

Wang

Chen

et al. 2021

Front. Cell. Infect. Microbiol.

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Metatranscriptomic data mining together with microfluidic card uncovered the potential pathogens and seasonal RNA viral ecology in a drinking water source

Shen,

Zhang,

Wang

et al. 2023

Journal of Applied Microbiology

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Aims Despite metatranscriptomics becoming an emerging tool for pathogen surveillance, very little is known about the feasibility of this approach for understanding the fate of human-derived pathogens in drinking water sources. Methods and results We conducted multiplexed microfluidic cards and metatranscriptomic sequencing of the drinking water source in a border city of North Korea in four seasons. Microfluidic card detected norovirus, hepatitis B virus (HBV), enterovirus, and Vibrio cholerae in the water. Phylogenetic analyses showed that environmental-derived sequences from norovirus GII.17, genotype C of HBV, and coxsackievirus A6 (CA6) were genetically related to the local clinical isolates. Meanwhile, metatranscriptomic assembly suggested that several bacterial pathogens, including Acinetobacter johnsonii and V. cholerae might be prevalent in the studied region. Metatranscriptomic analysis recovered 349 species-level groups with substantial viral diversity without detection of norovirus, HBV, and CA6. Seasonally distinct virus communities were also found. Specifically, 126, 73, 126, and 457 types of viruses were identified in spring, summer, autumn, and winter, respectively. The viromes were dominated by the Pisuviricota phylum, including members from Marnaviridae, Dicistroviridae, Luteoviridae, Potyviridae, Picornaviridae, Astroviridae, and Picobirnaviridae families. Further phylogenetic analyses of RNA (Ribonucleic Acid)-dependent RNA polymerase (RdRp) sequences showed a diverse set of picorna-like viruses associated with shellfish, of which several novel picorna-like viruses were also identified. Additionally, potential animal pathogens, including infectious bronchitis virus, Bat dicibavirus, Bat nodavirus, Bat picornavirus 2, infectious bursal disease virus, and Macrobrachium rosenbergii nodavirus were also identified. Conclusions Our data illustrate the divergence between microfluidic cards and metatranscriptomics, highlighting that the combination of both methods facilitates the source tracking of human viruses in challenging settings without sufficient clinical surveillance.

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Rapid and Visual Detection of Actinidia Chlorotic Ringspot-Associated Virus Using One-Step Reverse-Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay

et al. 2023

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Actinidia chlorotic ringspot-associated virus (AcCRaV) occurs widely in major kiwifruit producing areas of China and is often accompanied by co-infecting viruses, affecting the growth, yield and quality of kiwifruit. Therefore, a rapid and sensitive detection method is crucial for the diagnosis, control as well as eradication of AcCRaV. In this study, a one-step reverse transcription-recombinase polymerase amplification combined with a lateral flow dipstick (RT-RPA-LFD) assay was developed for rapid detection of AcCRaV. Specific primers and a probe were designed based on the conserved region of the coat protein gene sequence of AcCRaV. The one-step RT-RPA reaction can be performed at 35 °C and 40 °C within 10 to 30 min, and the amplification results can be read directly on the LFD within 5 min. The detection limits of the one-step RT-RPA-LFD assay were 10-8 ng (about 20 viral copies), which was equal with one-step RT-qPCR and 100 times more sensitive than one-step RT-PCR. Moreover, the one-step RT-RPA-LFD assay was successfully applied to detect AcCRaV from crude extracts, and the entire detection process can be completed within 40 min. These results indicate that the RT-RPA-LFD assay is a simple, rapid and sensitive strategy that can be used for rapid diagnosis of AcCRaV-infected kiwifruit plants in the field. To our knowledge, this is the first study applying one-step RT-RPA-LFD assay to detect kiwifruit virus.

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One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

Cited by 5 publications

References 30 publications

Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip

Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip

Metatranscriptomic data mining together with microfluidic card uncovered the potential pathogens and seasonal RNA viral ecology in a drinking water source

Rapid and Visual Detection of Actinidia Chlorotic Ringspot-Associated Virus Using One-Step Reverse-Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay

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