National Academy of Agricultural Science, Rural Development Administration, 441-707 Suwon, Republic of Korea.
Appl Microbiol Biotechnol. 2013 Apr;97(8):3643-51. doi: 10.1007/s00253-013-4709-0. Epub 2013 Mar 17.
The aim of the present study was to develop a noble and specific marker for a quantitative polymerase chain reaction (PCR) assay for the species-specific detection of Pseudomonas aeruginosa based on the O-antigen acetylase gene. It is an important challenge to characterize populations of the bacterium P. aeruginosa, an opportunist by virtue of its physiological and genetic adaptability. However, molecular and serological methods currently available for sensitive and specific detection of P. aeruginosa are by no means satisfactory because there have been critical defects in the diagnosis and identification of P. aeruginosa strains in that these assays also detect other Pseudomonas species, or do not obtain amplified products from P. aeruginosa strains. Therefore, a primer set was designed based on the O-antigen acetylase gene of P. aeruginosa PA01 because it has been known that this gene is structurally diverse among species. The specificity of the primer set was evaluated using genomic DNA from six isolates of P. aeruginosa, 18 different species of Pseudomonas, and 23 other reference pathogenic bacteria. The primer set used in the PCR assay amplified a 232-bp amplicon for only six P. aeruginosa strains. The assay was also able to detect at least 1.41 × 10(3) copies/μl of cloned amplified target DNA using purified DNA, or 2.7 × 10(2) colony-forming unit per reaction when using calibrated cell suspension. In conclusion, this assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of water with a low level or latent infection of P. aeruginosa.
本研究旨在开发一种基于 O 抗原乙酰酶基因的聚合酶链反应（PCR）定量检测铜绿假单胞菌的特异性标志物。由于其生理和遗传适应性，铜绿假单胞菌种群的特征描述是一项重要的挑战。然而，目前可用于敏感和特异性检测铜绿假单胞菌的分子和血清学方法并不令人满意，因为在铜绿假单胞菌菌株的诊断和鉴定方面存在关键缺陷，这些检测方法也会检测到其他假单胞菌属的细菌，或者不能从铜绿假单胞菌菌株中获得扩增产物。因此，根据铜绿假单胞菌 PA01 的 O 抗原乙酰酶基因设计了一组引物，因为已知该基因在物种间具有结构多样性。使用来自 6 株铜绿假单胞菌、18 种不同的假单胞菌和 23 种其他参考病原菌的基因组 DNA 评估了引物组的特异性。PCR 检测仅针对 6 株铜绿假单胞菌扩增出 232bp 的扩增子。当使用纯化的 DNA 时，该检测方法也能够检测到至少 1.41×10(3)拷贝/μl 的克隆扩增靶 DNA，而当使用校准的细胞悬浮液时，每个反应可检测到 2.7×10(2)菌落形成单位。总之，该检测方法可作为一种实用的诊断方法，用于流行病学研究和低水平或潜伏性铜绿假单胞菌感染水的卫生管理。
