Improved PCR for identification of Pseudomonas aeruginosa

Abstract

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.

Detection of the major bacterial pathogens among children suffering from empyema in Ahvaz city, Iran

“…The molecular identifications of the major bacterial pathogens were performed by the amplifications of species‐specific genes. The PCR targets were the lytA gene for S pneumoniae , the P1 gene for H influenzae , the femA gene for S aureus , and the ecfX gene for P aeruginosa . The primer sets used for the identification of P aeruginosa , S pneumoniae , H influenzae, and S aureus and their annealing temperatures are listed in Table .…”

Methodological approaches for monitoring opportunistic pathogens in premise plumbing: A review

“…However, these assays have been criticized for false positive and/or false-negative detection of P. aeruginosa , likely owing to wide sequences variation of P. aeruginosa strains and frequent genetic exchange with other microorganisms (Anuj et al, 2009; Choi et al, 2013; Qin et al, 2003; Schwartz et al, 2006). As a result, some researchers have proposed that a combination of two targets is more suitable for increased specificity and sensitivity of P. aeruginosa identification (Anuj et al, 2009; Qin et al, 2003).…”

“…Choi et al recently took advantage of comparative genomic tools and developed a new q-PCR assay targeting the O-antigen acetylase gene. The specificity of this assay was tested against 6 P. aeruginosa isolates, 18 different Pseudomonas species and 23 other reference pathogens (Choi et al, 2013). However, tests against environmental samples would be needed for further assay specificity validation considering variation of background microorganisms in different sample matrices.…”

Exacerbation of bronchiectasis by Pseudomonas monteilii: a case report

“…It was identified as a colonizer in pharyngeal and rectal swabs collected from Syrian refugees, including a meropenem resistant isolate [11]. This organism is also a potential MBL reservoir, with environmental strains having been found to harbour bla VIM-13 and bla VIM-2 genes [3][4][5][6][7][8][9][10][11][12].…”

“…Two primers targeting different genes which have been used to identify P. aeruginosa were employed [5,6]. Two isolates, one clinical and one environmental, tested positive for the O-Antigen acetylase gene but not for 16S rDNA v2 and v8.…”