Detection of Pseudomonas aeruginosa by a triplex polymerase chain reaction assay based on lasI/R and gyrB genes

Abstract

Pseudomonas aeruginosa is a nosocomial pathogen, which, due to its inherent and acquired resistance to a wide range of antibiotics, causes high mortality rates. Therefore, rapid detection of the bacterium with high specificity and sensitivity plays a critical role in the control of the pathogenic bacterium. The aim of this study was to evaluate the accuracy and specificity of a prompt detection of the bacterium based on a triplex polymerase chain reaction that amplifies the lasI, lasR and gyrB genes. For this purpose, 30 clinical isolates of P. aeruginosa and 30 wound biopsy samples were retrieved from clinical diagnostic laboratories. After the extraction of the chromosomal DNA, the desired genes were amplified using uniplex and triplex PCR with appropriate primers. The specificity of the primers was evaluated by a comparison of the PCR results for P. aeruginosa clinical samples and non-Pseudomonas species control samples. The sensitivity of the primers was determined using a serial dilution of the genomic DNA template (100 ng to 100 fg) and by a comparison of the PCR and bacterial culture results. The results showed that the triplex PCR assay was positive for all of the samples (100%), while the PCR identifications were negative for non-Pseudomonas species. Additionally, at 10(-4) and 10(-5) diluted genomic DNA from P. aeruginosa (10 pg and 1 pg), the triplex PCR test was positive for the Las and gyrB genes in all of the samples, respectively. Based on these results, the designed primers can be used for the rapid, specific and sensitive diagnosis of P. aeruginosa in a triplex PCR assay.

Recent Advances in Molecular Diagnosis of Pseudomonasaeruginosa Infection by State-of-the-Art Genotyping Techniques

“…The primer–primer competition and relative abundance of target in regard to primer concentration need to be taken into consideration. To date, no standard protocol for the detection of P. aeruginosa using multiplex-PCR is available despite of the development and continuous improvement of this approach ( De Vos et al, 1997 ; da Silva Filho et al, 2004 ; Anuj et al, 2009 ; Thong et al, 2011 ; Salman et al, 2013 ; Aghamollaei et al, 2015 ).…”

Minimum Inhibitory Concentration of Ciprofloxacin against Pseudomonas Aeruginosa in the Presence of the Efflux Inhibitor Phenylalanine-arginine Beta-naphthylamide

“…Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative opportunistic pathogen causing infection in patients with a compromised host defense system such as burn injuries, acute keratitis, and cystic fibrosis (1). The treatment of infections in hospitalized patients is difficult due to the presence of antibiotic-resistant isolates (2). P. aeruginosa is the leading cause of death among people with cystic fibrosis (3).…”

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“…Generally, the transcription of many pathogenesis-related RNAs is dependent on the growth phase but in several pathogens, the secretion of virulence factors is regulated by cell-density sensing (quorum sensing), a process that involves communication through secreted signaling molecules. Several regulatory RNAs are the main effectors of quorum-sensing systems [38, 39]. …”