Please use this identifier to cite or link to this item:
Title: Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa
Authors: Manajit O.
Longyant S.
Sithigorngul P.
Chaivisuthangkura P.
Keywords: DNA polymerase
gold nanoparticle
thymidine triphosphate
uracil DNA glycosidase
metal nanoparticle
uracil DNA glycosidase
bacterium contamination
bacterium detection
bacterium identification
colony forming unit
controlled study
cross reaction
DNA hybridization
ecfx gene
limit of detection
loop mediated isothermal amplification
nucleic acid amplification
Pseudomonas aeruginosa
sensitivity and specificity
molecular probe
polymerase chain reaction
Pseudomonas aeruginosa
Pseudomonas infection
Metal Nanoparticles
Molecular Probes
Nucleic Acid Amplification Techniques
Polymerase Chain Reaction
Pseudomonas aeruginosa
Pseudomonas Infections
RNA, Ribosomal, 16S
Sensitivity and Specificity
Uracil-DNA Glycosidase
Issue Date: 2018
Abstract: Pseudomonas aeruginosa (P. aeruginosa) is an important opportunistic pathogen that causes serious infections in humans, including keratitis in contact lens wearers. Therefore, establishing a rapid, specific and sensitive method for the identification of P. aeruginosa is imperative. In the present study, the uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification combined with nanogold labeled hybridization probe (UDG-LAMP-AuNP) was developed for the detection of P. aeruginosa. UDG-LAMP was performed to prevent carry over contamination and the LAMP reactions can be readily observed using the nanogold probe. A set of 4 primers and a hybridization probe were designed based on the ecfX gene. The UDG-LAMP reactions were performed at 65°C for 60 min using the ratio of 40% deoxyuridine triphosphate to 60% deoxythymidine triphosphate. The detection of UDG-LAMP products using the nanogold labeled hybridization probe, which appeared as a red-purple color, was examined at 65°C for 5 min with 40 mM MgSO4. The UDG-LAMP-AuNP demonstrated specificity to all tested isolates of P. aeruginosa without cross reaction to other bacteria. The sensitivity for the detection of pure culture was 1.6×103 colony-forming units (CFU) ml-1 or equivalent to 3 CFU per reaction while that of polymerase chain reaction was 30 CFU per reaction. The detection limit of spiked contact lenses was 1.1×103 CFU ml-1 or equivalent to 2 CFU per reaction. In conclusion, the UDG-LAMP-AuNP assay was rapid, simple, specific and was effective for the identification of P. aeruginosa in contaminated samples. © 2018 Kashyap.
ISSN: 17912997
Appears in Collections:Scopus 1983-2021

Files in This Item:
There are no files associated with this item.

Items in SWU repository are protected by copyright, with all rights reserved, unless otherwise indicated.