scholarly journals Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification

PLoS ONE ◽  
2014 ◽  
Vol 9 (11) ◽  
pp. e111874 ◽  
Author(s):  
Anja Mezger ◽  
Christina Öhrmalm ◽  
David Herthnek ◽  
Jonas Blomberg ◽  
Mats Nilsson
Keyword(s):  
Rolling Circle Amplification ◽  
Rolling Circle ◽  
Padlock Probes

scholarly journals Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza

2018 ◽  
Vol 64 (12) ◽  
pp. 1704-1712 ◽  
Author(s):  
Felix Neumann ◽  
Iván Hernández-Neuta ◽  
Malin Grabbe ◽  
Narayanan Madaboosi ◽  
Jan Albert ◽  
...  
Keyword(s):  
Seasonal Influenza ◽  
Rolling Circle Amplification ◽  
High Specificity ◽  
Clinical Samples ◽  
Padlock Probe ◽  
Rolling Circle ◽  
Diagnostic Assays ◽  
Padlock Probes ◽  
Probe Assay ◽  
Digital Quantification

Abstract BACKGROUND Influenza remains a constant threat worldwide, and WHO estimates that it affects 5% to 15% of the global population each season, with an associated 3 to 5 million severe cases and up to 500000 deaths. To limit the morbidity and the economic burden of influenza, improved diagnostic assays are needed. METHODS We developed a multiplexed assay for the detection and subtyping of seasonal influenza based on padlock probes and rolling circle amplification. The assay simultaneously targets all 8 genome segments of the 4 circulating influenza variants—A(H1N1), A(H3N2), B/Yamagata, and B/Victoria—and was combined with a prototype cartridge for inexpensive digital quantification. Characterized virus isolates and patient nasopharyngeal swabs were used for assay design and analytical validation. The diagnostic performance was assessed by blinded testing of 50 clinical samples analyzed in parallel with a commercial influenza assay, Simplexa™ Flu A/B & RSV Direct. RESULTS The assay had a detection limit of 18 viral RNA copies and achieved 100% analytical and clinical specificity for differential detection and subtyping of seasonal circulating influenza variants. The diagnostic sensitivity on the 50 clinical samples was 77.5% for detecting influenza and up to 73% for subtyping seasonal variants. CONCLUSIONS We have presented a proof-of-concept padlock probe assay combined with an inexpensive digital readout for the detection and subtyping of seasonal influenza strains A and B. The demonstrated high specificity and multiplexing capability, together with the digital quantification, established the assay as a promising diagnostic tool for seasonal influenza.

scholarly journals SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

2020 ◽  
Author(s):  
Alexandros Sountoulidis ◽  
Andreas Liontos ◽  
Hong Phuong Nguyen ◽  
Alexandra B. Firsova ◽  
Athanasios Fysikopoulos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rolling Circle Amplification ◽  
Marker Gene ◽  
Cell Types ◽  
Experimental Conditions ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Tissue Sections ◽  
Specificity And Sensitivity

AbstractChanges in cell identities and positions underlie tissue development and disease progression. Although, single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell-states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single Cell Resolution INSitu Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRISHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity and quantitative qualities of SCRINSHOT facilitate single cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

scholarly journals SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000675
Author(s):  
Alexandros Sountoulidis ◽  
Andreas Liontos ◽  
Hong Phuong Nguyen ◽  
Alexandra B. Firsova ◽  
Athanasios Fysikopoulos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rolling Circle Amplification ◽  
Marker Gene ◽  
Cell Types ◽  
Experimental Conditions ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Tissue Sections ◽  
Specificity And Sensitivity

Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.

scholarly journals In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes

2004 ◽  
Vol 1 (3) ◽  
pp. 227-232 ◽  
Author(s):  
Chatarina Larsson ◽  
Jørn Koch ◽  
Anders Nygren ◽  
George Janssen ◽  
Anton K Raap ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Dna Molecules ◽  
Rolling Circle ◽  
Padlock Probes

scholarly journals Environmental Screening of Fonsecaea Agents of Chromoblastomycosis Using Rolling Circle Amplification

2020 ◽  
Vol 6 (4) ◽  
pp. 290
Author(s):  
Morgana Ferreira Voidaleski ◽  
Renata Rodrigues Gomes ◽  
Conceição de Maria Pedrozo e Silva de Azevedo ◽  
Bruna Jacomel Favoreto de Souza Lima ◽  
Flávia de Fátima Costa ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Rolling Circle Amplification ◽  
Low Frequency ◽  
Plant Materials ◽  
Fungal Isolation ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Endemic Areas ◽  
Tissue Implantation ◽  
Tropical Climates

Chromoblastomycosis is a chronic, cutaneous or subcutaneous mycosis characterized by the presence of muriform cells in host tissue. Implantation disease is caused by melanized fungi related to black yeasts, which, in humid tropical climates, are mainly members of the genus Fonsecaea. In endemic areas of Brazil, F. pedrosoi and F. monophora are the prevalent species. The current hypothesis of infection is traumatic introduction via plant materials, especially by plant thorns. However, isolation studies have demonstrated a low frequency of the agents in environmental substrates. The present study aimed to detect F. pedrosoi and F. monophora in shells of babassu coconuts, soil, plant debris, and thorns from endemic areas of chromoblastomycosis in Maranhão state, northern Brazil, using Rolling Circle Amplification (RCA) with padlock probes as a new environmental screening tool for agents of chromoblastomycosis. In addition to molecular screening, the environmental samples were analyzed by fungal isolation using mineral oil flotation. The limit of detection of the RCA method was 2.88 × 107 copies of DNA per sample for the used padlock probes, indicating that this represents an efficient and sensitive molecular tool for the environmental screening of Fonsecaea agents. In contrast, with isolation from the same samples using several selective methods, no agents of chromoblastomycosis were recovered.

Molecular identification of Neofabraea species associated with bull’s-eye rot on apple using rolling-circle amplification of partial EF-1α sequence

2018 ◽  
Vol 64 (1) ◽  
pp. 57-68 ◽  
Author(s):  
Huijiao Lin ◽  
Xiang Jiang ◽  
Jianping Yi ◽  
Xinguo Wang ◽  
Ranling Zuo ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Pathogenic Fungi ◽  
Epidemiological Studies ◽  
Rapid Identification ◽  
Target Species ◽  
Probe Signal ◽  
Closely Related Species ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Bull's Eye

A rolling-circle amplification (RCA) method with padlock probes targeted on EF-1α regions was developed for rapid detection of apple bull’s-eye rot pathogens, including Neofabraea malicorticis, N. perennans, N. kienholzii, and N. vagabunda (synonym: N. alba). Four padlock probes (PLP-Nm, PLP-Np, PLP-Nk, and PLP-Nv) were designed and tested against 28 samples, including 22 BER pathogen cultures, 4 closely related species, and 2 unrelated species that may cause serious apple decays. The assay successfully identified all the bull’s-eye rot pathogenic fungi at the level of species, while no cross-reaction was observed in all target species and no false-positive reaction was observed with all strains used for reference. This study showed that the use of padlock probes and the combination of probe signal amplification by RCA provided an effective and sensitive method for the rapid identification of Neofabraea spp. The method could therefore be a useful tool for monitoring bull’s-eye rot pathogens in port quarantine and orchard epidemiological studies.

scholarly journals Electrochemical Genosensing of E. coli Based on Padlock Probes and Rolling Circle Amplification

Sensors ◽  
2021 ◽  
Vol 21 (5) ◽  
pp. 1749
Author(s):  
Alejandra Ben Aissa ◽  
Narayanan Madaboosi ◽  
Mats Nilsson ◽  
Maria Isabel Pividori
Keyword(s):  
Pathogenic Bacteria ◽  
Magnetic Particles ◽  
Direct Detection ◽  
Rolling Circle Amplification ◽  
Isothermal Amplification ◽  
Bacterial Dna ◽  
Rolling Circle ◽  
E Coli ◽  
Padlock Probes ◽  
Reliable Power Supply

Isothermal amplification techniques are emerging nowadays for the rapid and accurate detection of pathogenic bacteria in low resource settings, where many infectious diseases are endemic, and the lack of reliable power supply, trained personnel and specialized facilities pose critical barriers for timely diagnosis. This work addresses the detection of E. coli based on DNA isothermal amplification performed on magnetic particles (MPs) followed by electrochemical genosensing on disposable electrodes by square-wave voltammetry. In this approach, the bacterial DNA is preconcentrated using a target-specific magnetic probe and then amplified on the MPs by rolling circle amplification (RCA). Two different electrochemical readout methods for the RCA amplicons are tested. The first one relied on the labelling of the magnetic RCA product with a digoxigenin probe followed by the incubation with antiDIG-HRP antibody as electrochemical reporter. In the second case, the direct detection with an HRP-probe was performed. This latter strategy showed an improved analytical performance, while simultaneously avoiding the use of thermocyclers or bulky bench top equipment.

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