title: 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.
authors: Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors: Hosseine
authors: H
authors: Aghamollaei
authors: Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. Electronic address: mm.genetics@gmail.com.
authors: Mehrdad M
authors: MM
authors: Moghaddam
authors: Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors: Hamid
authors: H
authors: Kooshki
authors: Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors: Mohammad
authors: M
authors: Heiat
authors: Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors: Reza
authors: R
authors: Mirnejad
authors: National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors: Nastaran S
authors: NS
authors: Barzi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D027081:DNA Gyrase / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D011552:Pseudomonas Infections / Q000175:diagnosis*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification*; D053038:Quorum Sensing / Q000502:physiology; D012680:Sensitivity and Specificity; D015534:Trans-Activators / Q000235:genetics*; D014947:Wounds and Injuries / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; C081237:LasI protein, Pseudomonas aeruginosa; C080365:LasR protein, Pseudomonas aeruginosa; D015534:Trans-Activators; D027081:DNA Gyrase
keywords: Detection; Pseudomonas aeruginosa; Quorum sensing genes; Triplex PCR; gyrB gene