Rolling circle amplification with electrochemical detection assay for SARS-CoV-2

Abstract This protocol describes the rolling circle amplification (RCA) and electrochemical detection of SARS-CoV-2. The procedure consists of 3 parts, which are the amplification, hybridization and detection steps. In the presence of RNA template, the circular DNA template will be ligated to produce a Padlock DNA, which serves as a template for amplification by phi29 DNA polymerase to produce RCA amplicons. The RCA amplicon is a concatemer containing multiple repeats of sequences that are complementary to the circular template. The RCA amplicons are hybridized with redox active probes and detected by electrochemical biosensor using differential pulse voltammetry (DPV). Due to its isothermal nature, RCA can be performed using a simple water bath or heating block. Overall, the whole assay takes approximately 45 min. The assay enables rapid, quantitative results to be obtained for detection of SARS-CoV-2, either in the laboratory or more importantly, in a field setting.

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MiRNA Detection Using a Rolling Circle Amplification and RNA-Cutting Allosteric Deoxyribozyme Dual Signal Amplification Strategy

Biosensors ◽

10.3390/bios11070222 ◽

2021 ◽

Vol 11 (7) ◽

pp. 222

Author(s):

Chenxin Fang ◽

Ping Ouyang ◽

Yuxing Yang ◽

Yang Qing ◽

Jialun Han ◽

...

Keyword(s):

Signal Amplification ◽

Rolling Circle Amplification ◽

Padlock Probe ◽

Rolling Circle ◽

Sensing Platform ◽

Mirna Detection ◽

Substrate Cleavage ◽

Amplification Strategy ◽

Circular Template ◽

Dual Signal Amplification

A microRNA (miRNA) detection platform composed of a rolling circle amplification (RCA) system and an allosteric deoxyribozyme system is proposed, which can detect miRNA-21 rapidly and efficiently. Padlock probe hybridization with the target miRNA is achieved through complementary base pairing and the padlock probe forms a closed circular template under the action of ligase; this circular template results in RCA. In the presence of DNA polymerase, RCA proceeds and a long chain with numerous repeating units is formed. In the presence of single-stranded DNA (H1 and H2), multi-component nucleic acid enzymes (MNAzymes) are formed that have the ability to cleave substrates. Finally, substrates containing fluorescent and quenching groups and magnesium ions are added to the system to activate the MNAzyme and the substrate cleavage reaction, thus achieving fluorescence intensity amplification. The RCA–MNAzyme system has dual signal amplification and presents a sensing platform that demonstrates broad prospects in the analysis and detection of nucleic acids.

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Rapid electrochemical detection of coronavirus SARS-CoV-2

Nature Communications ◽

10.1038/s41467-021-21121-7 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

Thanyarat Chaibun ◽

Jiratchaya Puenpa ◽

Tatchanun Ngamdee ◽

Nimaradee Boonapatcharoen ◽

Pornpat Athamanolap ◽

...

Keyword(s):

Electrochemical Biosensor ◽

Rolling Circle Amplification ◽

Clinical Samples ◽

Rolling Circle ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Sandwich Hybridization ◽

Redox Active ◽

One Step ◽

The One

AbstractCoronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.

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HaloTag mediated artificial cellulosome assembly on a rolling circle amplification DNA template for efficient cellulose hydrolysis

Chemical Communications ◽

10.1039/c6cc02035f ◽

2016 ◽

Vol 52 (40) ◽

pp. 6701-6704 ◽

Cited By ~ 14

Author(s):

Qing Sun ◽

Wilfred Chen

Keyword(s):

Rolling Circle Amplification ◽

Cellulose Hydrolysis ◽

The Self ◽

Rolling Circle ◽

Dna Template ◽

Dna Scaffold

We report here the generation of artificial cellulosomes onto a DNA scaffold using the self-labeling HaloTag for DNA conjugation. Rolling circle amplification multiplexing templates were used to increase the complexity of this system with higher efficiency observed.

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Cascade Dual-Signal Enhancement by Integrating Exonuclease III-Assisted Target-Recycling and Rolling Circle Amplification for Ultrasensitive Electrochemical Detection of DNA

Journal of The Electrochemical Society ◽

10.1149/2.0511713jes ◽

2017 ◽

Vol 164 (13) ◽

pp. B603-B609 ◽

Cited By ~ 3

Author(s):

Xingxing Zhou ◽

Shijing Guo ◽

Jiaxi Gao ◽

Jianmin Zhao ◽

Wenju Xu

Keyword(s):

Electrochemical Detection ◽

Rolling Circle Amplification ◽

Signal Enhancement ◽

Exonuclease Iii ◽

Target Recycling ◽

Rolling Circle

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Aptamer-Based Rolling Circle Amplification: A Platform for Electrochemical Detection of Protein

Analytical Chemistry ◽

10.1021/ac071059s ◽

2007 ◽

Vol 79 (19) ◽

pp. 7492-7500 ◽

Cited By ~ 186

Author(s):

Long Zhou ◽

Li-Juan Ou ◽

Xia Chu ◽

Guo-Li Shen ◽

Ru-Qin Yu

Keyword(s):

Electrochemical Detection ◽

Rolling Circle Amplification ◽

Rolling Circle

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Rolling Circle Amplification as an Efficient Analytical Tool for Rapid Detection of Contaminants in Aqueous Environments

Biosensors ◽

10.3390/bios11100352 ◽

2021 ◽

Vol 11 (10) ◽

pp. 352

Author(s):

Kuankuan Zhang ◽

Hua Zhang ◽

Haorui Cao ◽

Yu Jiang ◽

Kang Mao ◽

...

Keyword(s):

Nucleic Acid ◽

Rolling Circle Amplification ◽

Environmental Pollutants ◽

Monitoring Method ◽

Rolling Circle ◽

Advantages And Disadvantages ◽

Contaminant Monitoring ◽

Aqueous Environments ◽

Global Concern ◽

Circular Template

Environmental contaminants are a global concern, and an effective strategy for remediation is to develop a rapid, on-site, and affordable monitoring method. However, this remains challenging, especially with regard to the detection of various contaminants in complex water environments. The application of molecular methods has recently attracted increasing attention; for example, rolling circle amplification (RCA) is an isothermal enzymatic process in which a short nucleic acid primer is amplified to form a long single-stranded nucleic acid using a circular template and special nucleic acid polymerases. Furthermore, this approach can be further engineered into a device for point-of-need monitoring of environmental pollutants. In this paper, we describe the fundamental principles of RCA and the advantages and disadvantages of RCA assays. Then, we discuss the recently developed RCA-based tools for environmental analysis to determine various targets, including heavy metals, organic small molecules, nucleic acids, peptides, proteins, and even microorganisms in aqueous environments. Finally, we summarize the challenges and outline strategies for the advancement of this technique for application in contaminant monitoring.

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Use of RCA-PCR assay for detection of Begomovirus infection in Bhut Jolokia (Capsicum assamicum) in Tezpur region of Assam, India

South Asian Journal of Experimental Biology ◽

10.38150/sajeb.6(2).p64-69 ◽

2016 ◽

Vol 6 (2) ◽

pp. 64-69

Author(s):

Ajitabh Bora ◽

Hemant Kumar Gogoi ◽

Mohan C. Kalita

Keyword(s):

Rolling Circle Amplification ◽

Viral Disease ◽

Viral Pathogen ◽

Rolling Circle ◽

North East India ◽

North East ◽

Detection Assay ◽

A Genome ◽

Bhut Jolokia ◽

Leaf Curl

Bhut Jolokia (Capsicum assamicum), one of the hottest chilli in the world is a chilli cultivar, endemic to North East India and is extensively cultivated in the states of Assam, Nagaland and Manipur. The demand of this chilli is very high in domestic as well as in the international market due to its extreme hotness and pleasant aroma. This plant is severely affected by leaf curl disease caused by Begomovirus, leading to total crop loss. Accurate diagnosis of this viral disease at seedling stage, prior to transplanting, is essential to prevent further spread of the disease. With an aim to device a rapid and accurate detection assay, Rolling Circle Amplification (RCA)-Polymerase Chain Reac-tion (PCR) technique was used for detection of the viral pathogen. RCA tech-nique was employed to increase the viral template and PCR was conducted using a degenerate primer pair. With the help of this assay, 1.4 kbp segment of DNA-A genome of the Begomovirus was amplified. Phylogeny revealed close proximity of the isolate with Tomato leaf curl Bangladesh virus. This is the first report on characterization of Begomovirus infecting Bhut Jolokia in Tezpur region of Assam.

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