Use of Padlock Probes and Rolling Circle Amplification (RCA) for Rapid Identification of Trichophyton Species, Related to Human and Animal Disorder

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.

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Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza

Clinical Chemistry ◽

10.1373/clinchem.2018.292979 ◽

2018 ◽

Vol 64 (12) ◽

pp. 1704-1712 ◽

Cited By ~ 10

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.

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Rolling Circle Amplification with Padlock Probes for In Situ Detection of RNA Analytes

Rolling Circle Amplification (RCA) ◽

10.1007/978-3-319-42226-8_9 ◽

2016 ◽

pp. 99-105

Author(s):

Anja Mezger ◽

Malte Kühnemund ◽

Mats Nilsson

Keyword(s):

Rolling Circle Amplification ◽

Rolling Circle ◽

Padlock Probes ◽

In Situ Detection

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Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification

PLoS ONE ◽

10.1371/journal.pone.0031068 ◽

2012 ◽

Vol 7 (2) ◽

pp. e31068 ◽

Cited By ~ 35

Author(s):

Jenny Göransson ◽

Rongqin Ke ◽

Rachel Yuan Nong ◽

W. Mathias Howell ◽

Anna Karman ◽

...

Keyword(s):

Rolling Circle Amplification ◽

Rapid Identification ◽

Rolling Circle

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A microRNA detection system based on padlock probes and rolling circle amplification

RNA ◽

10.1261/rna.110706 ◽

2006 ◽

Vol 12 (9) ◽

pp. 1747-1752 ◽

Cited By ~ 154

Author(s):

S. P. Jonstrup

Keyword(s):

Detection System ◽

Rolling Circle Amplification ◽

Rolling Circle ◽

Padlock Probes ◽

Microrna Detection

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

10.1101/2020.02.07.938571 ◽

2020 ◽

Cited By ~ 1

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.

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

PLoS Biology ◽

10.1371/journal.pbio.3000675 ◽

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.

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