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Colorimetric nucleic acid testing assay for RNA virus detection based on circle-to-circle amplification of padlock probes.

ABSTRACT: We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 10(3) viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.

SUBMITTER: Ke R

PROVIDER: S-EPMC3232973 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Colorimetric nucleic acid testing assay for RNA virus detection based on circle-to-circle amplification of padlock probes.

Ke Rongqin R Zorzet Anna A Göransson Jenny J Lindegren Gunnel G Sharifi-Mood Batool B Chinikar Sadegh S Mardani Masoud M Mirazimi Ali A Nilsson Mats M

Journal of clinical microbiology 20110928 12

We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 10(3) viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing differ ...[more]

PMID: 21956984

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Project description:BACKGROUND: Isothermal amplification methods provide alternatives to PCR that may be preferable for some nucleic acid target detection tasks. Among current isothermal target detection methods, ramified rolling circle amplification (RAM) of single-stranded DNA circles that are formed by ligation of linear DNA probes (C-probes or padlock probes) offers a unique target detection system by linked primers and a simple amplification system that is unconstrained by the target's sequence context. Earlier implementations of RAM-based target detection were reported to be limited by background noise, due in part to unligated C-probe in the amplification reaction. We show here that a target-detection system using a biotinylated target-capture probe together with automated bead-handling reduces or eliminates background amplification noise. We demonstrate the system's performance by detection of a single-nucleotide polymorphism in human genomic DNA. METHODOLOGY: Target detection by RAM entails hybridization and ligation of a C-probe, followed by amplification and RAM signal detection. We evaluated RAM target detection in genomic DNA using recognition of a human Factor V gene single nucleotide polymorphism (G1691A) as a model. Locus-specific C-probes were annealed and ligated to genomic DNAs that represent the 3 possible genotypes at this locus, then ligated C-probes were amplified by real time RAM. The majority of the steps in the assay were performed with a magnetic bead-based chemistry on an automated platform. We show that the specificity of C-probe ligation permits accurate genotyping of this polymorphism. The assay as described here eliminates some of the background noise previously described for C-probe ligation, RAM amplification assays. CONCLUSION: The methods and results presented here show that a combination of C-probe detection, automated sample processing, and isothermal RAM amplification provide a practical approach for detecting DNA targets in complex mixtures.

Dataset Information

Colorimetric nucleic acid testing assay for RNA virus detection based on circle-to-circle amplification of padlock probes.

Publications

Colorimetric nucleic acid testing assay for RNA virus detection based on circle-to-circle amplification of padlock probes.

Similar Datasets

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