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.

scholarly journals An Origami Paper-Based Device Printed with DNAzyme-Containing DNA Superstructures for Escherichia coli Detection

Micromachines ◽  
2019 ◽  
Vol 10 (8) ◽  
pp. 531 ◽  
Author(s):  
Yating Sun ◽  
Yangyang Chang ◽  
Qiang Zhang ◽  
Meng Liu
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Rolling Circle Amplification ◽  
Health And Safety ◽  
Turnaround Time ◽  
Rolling Circle ◽  
E Coli ◽  
Resource Limited ◽  
First Time ◽  
Public Health And Safety

Rapid detection of pathogenic bacteria is extremely important for public health and safety. Here, we describe for the first time an integrated origami paper-based analytical device (PAD) incorporating cell lysis, molecular recognition, amplification and visual detection of Escherichia coli (E. coli). The device features three components: paper for its ability to extract protein molecules nonspecifically from cells, DNA superstructures for their ability to immobilize RNA-cleaving DNAzymes (RCDs) but undergo target-induced RNA cleavage on paper, and isothermal rolling circle amplification (RCA) for its ability to amplify each cleavage event into repetitive sequence units that can be detected by naked eye. This device can achieve detection of E. coli K12 with a detection limit of as low as 103 CFU·mL−1 in a total turnaround time of 35 min. Furthermore, this device allowed the sensitive detection of E. coli in complex sample matrices such as juice and milk. Given that more specific RCDs can be evolved for diverse bacteria, the integrated PAD holds great potential for rapid, sensitive and highly selective detection of pathogenic bacteria in resource-limited settings.

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

Direct detection of circulating microRNAs in serum of cancer patients by coupling protein-facilitated specific enrichment and rolling circle amplification

2014 ◽  
Vol 50 (25) ◽  
pp. 3292-3295 ◽  
Author(s):  
Cheng-Yi Hong ◽  
Xian Chen ◽  
Juan Li ◽  
Jing-Hua Chen ◽  
Guonan Chen ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Direct Detection ◽  
Rolling Circle Amplification ◽  
Circulating Mirnas ◽  
Simple Method ◽  
Circulating Micrornas ◽  
Rolling Circle ◽  
Coupling Protein

A simple method for direct detection of circulating miRNAs in serum by coupling p19 protein-facilitated specific enrichment and RCA.

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 Integration of isothermal amplification with quantum dot-based fluorescence resonance energy transfer for simultaneous detection of multiple microRNAs

2018 ◽  
Vol 9 (18) ◽  
pp. 4258-4267 ◽  
Author(s):  
Juan Hu ◽  
Ming-hao Liu ◽  
Chun-yang Zhang
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Fluorescence Resonance Energy Transfer ◽  
Rolling Circle Amplification ◽  
Simultaneous Detection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Isothermal Amplification ◽  
Rolling Circle ◽  
Fluorescence Resonance

The integration of quantum dot-based fluorescence resonance energy transfer with rolling circle amplification enables simultaneous sensitive detection of multiple microRNAs.

scholarly journals Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

2005 ◽  
Vol 71 (12) ◽  
pp. 7933-7940 ◽  
Author(s):  
Fumito Maruyama ◽  
Takehiko Kenzaka ◽  
Nobuyasu Yamaguchi ◽  
Katsuji Tani ◽  
Masao Nasu
Keyword(s):  
Gene Sequence ◽  
Rolling Circle Amplification ◽  
Specific Gene ◽  
Toxin Gene ◽  
Dna Uptake ◽  
Fluorescent Intensity ◽  
Prolonged Incubation ◽  
Rolling Circle ◽  
E Coli

ABSTRACT Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.

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