ABSTRACT
The 16S rDNA specific primers were designed for rapid detection of Pseudomonas aeruginosa (PA) by the fluorescence quantitative PCR (FQ-PCR) assay, based upon multiple sequence alignment and phylogenetic tree analysis of the 16S rDNAs of over 20 bacteria. After extraction of PA genomic DNA, the target 16S rDNA fragment was amplified by PCR with specific primers, and used to construct recombinant pMDT-Pfr plasmid, the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay. Different concentrations of PA genomic DNA were detected by FQ-PCR in a 20microL of reaction system with SYBR Green I. At the same time, various genomic DNAs of Staphylococcus aureus, Salmonella typhi, Shigella flexneri, Proteus vulgaris, Staphylococcus epidermidis, Escherichia coli, and Mycobacterium tuberculosis were used as negative controls to confirm specificity of the FQ-PCR detection assay. Results demonstrated that the predicted amplified product of designed primers was of high homology only with PA 16S rDNA, and that sensitivity of the FQ-PCR assay was of 3.6pg/microL of bacterial DNA or (2.1 x 10(3) +/- 3.1 x 10(2)) copies/microL of 16S rDNA, accompanied with high specificity, and that the whole detection process including DNA extraction could be completed in about two hours. In contrast to traditional culture method, the FQ-PCR assay targeting 16S rDNA gene can be used to detect PA rapidly, which exhibits perfect application prospect in future.
Subject(s)
Base Sequence , DNA, Ribosomal , Genetics , Fluorescence , Molecular Sequence Data , Polymerase Chain Reaction , Methods , Pseudomonas aeruginosa , Genetics , RNA, Ribosomal, 16S , GeneticsABSTRACT
One hundred Pseudomonas aeruginosa quorum-sensing-related genes were selected and their primers were synthesized. The fragments of specific sequences which are related QS genes were amplified by PCR. These verified sequences were inserted into the vector pMD-18T for sequencing. These DNA fragments were dotted onto glass slides to make cDNA microarray. Hybridization was performed with cy3/cy5-dCTP labeled probes. The scanning data of early stationary phase and mid-logarithmic phase indicated that 9 genes were up-regulated and 6 genes were down-regulated. Undergoing the different medicines,we took tobramycin as an example to compare the expression diversity. The results confirm that the QS cDNA chip is useful,and may contribute to better understand the mechanism of quorum-sensing,and can help us find the new targets for restraining the growth of Pseudomonas aeruginosa.
