Multiplex PCR strategy for characterization of modular genomic structure of Staphylococcus aureus bacteriophages
| Authors | |
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| Year of publication | 2008 |
| Type | Article in Proceedings |
| Conference | Pathophysiology of Staphylococci |
| MU Faculty or unit | |
| Citation | |
| Web | http://www.pathostaph.de/ |
| Field | Genetics and molecular biology |
| Keywords | Staphylococcus aureus; bacteriophages; molecular diagnostics; mobile genetic elements |
| Description | Pathogenic Staphylococcus aurues strains differ in the presence of virulence factors that are encoded mainly by mobile genetic elements, in particular by prophages. The study objective was to develop a method for rapid and simple characterization of S. aureus prophages. The prophages were induced from lysogenic strains by UV-irradiation. Phages were picked up from one plaque and propagated on a non-lysogenic strain to obtain a low titre phage lysate (10 e3 PFU/ml). A new method for phage DNA extraction from small volumes of low titre phage lysate was developed using magnetic nonporous microspheres P(HEMA-co-EDMA) and NucleoMag. The phage DNAs were characterized by multiplex PCR assays targeting capsid genes (portal and tail), genes for phage integrases, anti-repressors, amidases and virulence associated genes for Panton-Valentine leukocidin, exfoliative toxin A and those of innate immune evasion cluster. The PCR-ready DNA was isolated using novel method and amplified by PCR using newly designed primer sets. The results enabled us to divide the phage genomic modules into several types (numbers in brackets): capsid structure (9), integrases dictating the attachment site on the host chromosome (10), anti-repressor (10), and lytic module (4). We propose updating the phage nomenclature to correspond better to the genomic loci and extensive mosaic pattern of phage genomes. The rapid and simple method for DNA extraction followed by PCR based diagnosis of phage genomic modules is helpful in effective study of phage dynamics. |
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