Isolation of PCR-ready DNA from lyophilisates of <I>Trichophyton</I> fungi and from phages of <I>Staphylococcus aureus</I> using magnetic microspheres
| Authors | |
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| Year of publication | 2008 |
| Type | Article in Proceedings |
| Conference | 10 th International Symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers & 10 th International Symposium on Advances in Extraction Techniques 2008 |
| MU Faculty or unit | |
| Citation | |
| Web | http://www.ordibo.be/htc/ |
| Field | Genetics and molecular biology |
| Keywords | fungi; Trichophyton; bacteriophages; Staphylococcus aureus; nonporous magnetic hydrophilic microspheres; P(HEMA-co-EDMA) |
| Description | The aim of this contribution was focused on the isolation of PCR-ready DNA from Trichophyton lyophilised cells (spores) and from the small volumes of Staphylococcus aureus phage lysates. Extraction of PCR-ready DNA from microscopic fungi is crucial in dermatophytes due to the presence of fungal polysaccharides and the chitinous cell wall. PCR-ready DNA isolated from crude cell lysates was repurified by magnetic carriers containing carboxyl groups on the surface. The adsorbed DNA was eluted by TE buffer. The DNA in the eluate was used as a matrix in PCR amplification. ITS4 and ITS5 primers were used for the amplification of Trichophyton DNA. The PCR-ready DNA can be isolated from lyophilisates without cultivation of cells (spores) and the influence of PCR inhibitors which are present in lyophilisation media was eliminated. The high-titre (109 particles per ml) and low-titre (103 particles per ml) phage lysates of Staphylococcus aureus bacteriophages f77 and f53 were used for sample preparation and DNA isolation. The phage lysates were treated by proteinase K. The DNA extracted using magnetic microspheres was RNA-free and multiplex PCR was used for phage DNA identification. |
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