Ultrasensitive Real-Time Rolling Circle Amplification Detection Enhanced by Nicking-Induced Tandem-Acting Polymerases
- 27 June 2019
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 91 (15), 10102-10109
- https://doi.org/10.1021/acs.analchem.9b02073
Abstract
Padlock probe ligation-based rolling circle amplification (RCA) can distinguish single-nucleotide variants, which is promising for the detection of drug-resistance mutations in, e.g., Mycobacterium tuberculosis (Mtb). However, the clinical application of conventional linear RCA is restricted by its unsatisfactory picomolar-level limit of detection (LOD). Herein, we demonstrate the mechanism of a nicking-enhanced RCA (NickRCA) strategy that allows several polymerases to act simultaneously on the same looped template, generating single-stranded amplicon monomers. Limiting factors of NickRCA are investigated and controlled for higher amplification efficiency. Thereafter, we describe a NickRCA-based magnetic nanoparticle (MNP) dimer formation strategy combined with a real-time optomagnetic sensor monitoring MNP dimers. The proposed methodology is applied for the detection of a common Mtb rifampicin-resistance mutation, rpoB 531 (TCG/TTG). Without additional operation steps, an LOD of 15 fM target DNA is achieved with a total assay time of ca. 100 min. Moreover, the proposed biosensor holds the advantages of single-nucleotide mutation discrimination and the robustness to quantify targets in 10% serum samples. NickRCA produces short single-stranded monomers instead of the DNA coils produced in conventional RCA, which makes it more convenient for downstream operation, immobilization or detection, thus being applicable with different molecular tools and biosensors.Keywords
Funding Information
- Det Frie Forskningsr?d (4184-00121B)
- H2020 Marie Sklodowska-Curie Actions (713683)
- Milj?styrelsen (MST-141-01415)
This publication has 40 references indexed in Scilit:
- A DNAzyme Feedback Amplification Strategy for BiosensingAngewandte Chemie-International Edition, 2017
- Isothermal Amplification of Nucleic AcidsChemical Reviews, 2015
- Advances in isothermal amplification: novel strategies inspired by biological processesBiosensors and Bioelectronics, 2015
- Bioanalytical applications of isothermal nucleic acid amplification techniquesAnalytica Chimica Acta, 2015
- Rolling circle amplification: a versatile tool for chemical biology, materials science and medicineChemical Society Reviews, 2014
- Rolling Circle Amplification: Applications in Nanotechnology and Biodetection with Functional Nucleic AcidsAngewandte Chemie-International Edition, 2008
- Enhanced detection and distinction of RNA by enzymatic probe ligationNature Biotechnology, 2000
- Signal amplification of padlock probes by rolling circle replicationNucleic Acids Research, 1998
- Mutation detection and single-molecule counting using isothermal rolling-circle amplificationNature Genetics, 1998
- Padlock Probes: Circularizing Oligonucleotides for Localized DNA DetectionScience, 1994