Publication:
Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

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Issued Date

2018

Resource Type

Article

File Type

application/pdf

ISSN

17912997

DOI

10.3892/mmr.2018.8557

Other identifier(s)

2-s2.0-85043294357

Rights Holder(s)

มหาวิทยาลัยศรีนครินทรวิโรฒ

Bibliographic Citation

Molecular Medicine Reports. Vol 17, No.4 (2018), p.5734-5743

Suggested Citation

Manajit O., Longyant S., Sithigorngul P., Chaivisuthangkura P. Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa. Molecular Medicine Reports. Vol 17, No.4 (2018), p.5734-5743. doi:10.3892/mmr.2018.8557 Retrieved from: https://hdl.handle.net/20.500.14740/5981

Title

Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

Author(s)

Manajit O.
 Longyant S.
 Sithigorngul P.
 Chaivisuthangkura P.

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.

Subject(s)

DNA polymerase
 Gold nanoparticle
 RNA 16S
 Thymidine triphosphate
 Uracil DNA glycosidase
 Gold
 Metal nanoparticle
 RNA 16S
 Uracil DNA glycosidase
 Amplicon
 Article
 Bacterium contamination
 Bacterium detection
 Bacterium identification
 Colony forming unit
 Colorimetry
 Controlled study
 Cross reaction
 DNA hybridization
 Ecfx gene
 Gene
 Limit of detection
 Loop mediated isothermal amplification
 Nonhuman
 Nucleic acid amplification
 Pseudomonas aeruginosa
 Sensitivity and specificity
 Genetics
 Human
 Microbiology
 Molecular probe
 Polymerase chain reaction
 Procedures
 Pseudomonas aeruginosa
 Pseudomonas infection
 Gold
 Humans
 Metal Nanoparticles
 Molecular Probes
 Nucleic Acid Amplification Techniques
 Polymerase Chain Reaction
 Pseudomonas aeruginosa
 Pseudomonas Infections
 RNA, Ribosomal, 16S
 Sensitivity and Specificity
 Uracil-DNA Glycosidase

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URI

https://hdl.handle.net/20.500.14740/5981

Collections

Scopus: Year 1983-2021

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