[Screening and real-time quantitative detection for genomic signatures of Bacillus anthracis].

A total amount of 117 candidate chromosomal sequences were screened for the genomic signatures of Bacillus anthracis by a 2-step approach. Out of them, 19 genomic signatures sequences were selected, among which, 6 genomic signatures were competent as the target sequence of TaqMan real-time PCR method. With the most significant alignment and specificity, fragment C04, together with pagA gene and capB gene from virulence plasmids pX01 and pX02, was selected to establish a TaqMan real-time PCR assay. For each target sequence, as little as 10 to 100 copies per reaction could be detected. Twelve bacterial species including 7 from Bacillus cereus group which were closely related to Bacillus anthracis were used to test the specificity of this assay. Data revealed that the assay gained a 100% specificity. Further performance of the assay was assessed with 10 simulative contaminated environmental samples and 20 negative control environmental samples; all of the Bacillus anthracis contaminated samples gave strong positive signals while the control samples were negative. This specific and sensitive real-time PCR assay could be used in rapid confirmation or exclusion of potential bio-attacks and the diagnosis of anthrax.

[Rapid detection of Pseudomonas aeruginosa by the fluorescence quantitative PCR assay targeting 16S rDNA].

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.