期刊
MOLECULAR MEDICINE REPORTS
卷 17, 期 4, 页码 5734-5743出版社
SPANDIDOS PUBL LTD
DOI: 10.3892/mmr.2018.8557
关键词
Pseudomonas aeruginosa; loop-mediated isothermal amplification; uracil-DNA-glycosylase; gold nanoparticle
资金
- Higher Education Research Promotion, Office of the Higher Education Commission, Thailand
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 degrees 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 degrees 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 1.6x10(3) 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 1.1x10(3) 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.
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