期刊
ANALYTICAL BIOCHEMISTRY
卷 598, 期 -, 页码 -出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2020.113705
关键词
Karlodinium armiger; Recombinase polymerase amplification (RPA); Modified primers; Electrochemical detection; Enzyme labelled amplicons
资金
- Spanish Ministerio de Economia y Competitividad [CIGUASENSING BIO2017-87946-C2-1-R, SEASENSING BIO2014-56024-C2-1]
Genosensors for the detection of DNA via hybridisation normally require post-amplification processing such as the generation of single-stranded DNA and pre-detection labelling, complicating and lengthening the assay. A straightforward electrochemical genosensor, for the direct detection of isothermally generated nucleic acid amplicons via hybridisation is reported. The detection of Karlodinium armiger, responsible for harmful algae blooms was used as a model system to demonstrate the proof of concept. The approach exploits the use of specifically modified primers designed to generate amplicons with a central duplex flanked by a single-stranded tail at one end of the duplex and a horse-radish peroxidase on the other end. Individual gold electrodes of an array were functionalised with self-assembled monolayers of short thiolated DNA probes, designed to hybridise with the single-stranded tailed amplicon with the reporter enzyme label incorporated. The optimum amplification time was determined to be 60 min, at a fixed temperature of 37 degrees C. The hybridisation time to the enzyme labelled amplicon was optimised to be 10 min, but 2 min hybridisation time was also adequate. In this first example of using horse radish peroxidase-labelled primer in solution-phase recombinase polymerase amplification for subsequent detection via solid-phase hybridisation, the detection limit achieved was 0.4 fM, equivalent to 27622 cells/L, and the developed genosensor was applied to the detection of synthetic as well as genomic DNA, which had been extracted from a seawater sample.
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