Isothermal and rapid detection of pathogenic microorganisms using a nano-rolling circle amplification-surface plasmon resonance biosensor

Rolling circle amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogens without the need for sequencing. In this study, a surface plasmon resonance DNA biosensor based on RCA with a gold (Au) nanoparticle surface was established for isothermal ide...

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Bibliographic Details
Published in:Biosensors & bioelectronics 2014-12, Vol.62, p.280-287
Main Authors: Shi, Dachuan, Huang, Junfu, Chuai, Zhengran, Chen, Dong, Zhu, Xiaoyan, Wang, Huan, Peng, Jia, Wu, Haiyan, Huang, Qing, Fu, Weiling
Format: Article
Language:English
Subjects:
Au nanoparticles
Bacteria
Bacteria - genetics
Bacteria - isolation & purification
Bacteria - pathogenicity
Base Sequence
Biological and medical sciences
Biosensors
Biotechnology
Deoxyribonucleic acid
DNA, Bacterial - analysis
DNA, Bacterial - genetics
Fundamental and applied biological sciences. Psychology
Gene sequencing
Gold
Humans
Locks
Metal Nanoparticles
Methods. Procedures. Technologies
Molecular Sequence Data
Multiplex detection
Nanostructure
Nucleic Acid Amplification Techniques
Nucleic Acid Hybridization
Oligonucleotide Probes - genetics
Padlock probe
Pathogen
Plasmons
Rolling circle amplification
Surface plasmon resonance
Surface Plasmon Resonance - methods
Various methods and equipments
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Summary:Rolling circle amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogens without the need for sequencing. In this study, a surface plasmon resonance DNA biosensor based on RCA with a gold (Au) nanoparticle surface was established for isothermal identification of DNA. The probes included a specific padlock probe, a capture probe (CP), which is bound to biotin, and an Au nanoparticle-modified probe, which hybridizes with the RCA products. The CP was assembled on gold nanoparticles to increase its ability to bind and hybridize. The linear padlock probe, which was designed to circularize by ligation upon recognition of the bacterial pathogen-specific sequence in 16S rDNA, hybridizes to fully complementary sequences within the CP. Upon recognition, each target gene DNA is distinguished by localization onto the corresponding channel on the chip surface. Then, the immobilized CPs act as primers to begin the in situ solid-phase RCA reaction, which produces long single-stranded DNA. The RCA products fixed on the chip surface cause significant surface plasmon resonance angle changes. We demonstrated that six different bacterial pathogens can be identified simultaneously and that 0.5pM of synthetic oligonucleotides and 0.5pgμl−1 of genomic DNA from clinical samples can be detected by this method with low background signals. Therefore, the multiplex diagnostic method provides a highly sensitive and specific approach for the rapid identification of positive samples. •A new application of SPR biosensor based on RCA, which can measure six different pathogens.•The new method for the multiplex detection of pathogens was applied to clinical samples.•The RCA was combined with gold nanoparticles conjugated with DNA probes for signal amplification.•The signal amplification processes were monitored in real-time.
ISSN:0956-5663
1873-4235
DOI:10.1016/j.bios.2014.06.066