The Nano-gold Gene Chip Technology For Rapid Identification Of Common Superficial Fungi

ObjectiveThe common superficial fungi which isolated from clinical sample cultured bySabouraud’s medium. The ITS region of fungi was amplified by PCR, for this regionspecific oligonucleotide probes were designed.MethodsDNA was extracted from standard strains, superficial fungi strains and negativecontrol strains by the Biospin fungal gene group of DNA extraction kits. The internaltranscribed spacer(ITS) was amplified by fungal universal primers ITSl (biotin marked)and ITS4. Oligonucleotide probes targeting to the ITS1or ITS2of rDNA were designedby Array Designer4software, then candidate probes were submitted to GenBank forSpecificity analysis.ResultsThe fungal DNA PCR products were detected by agarose gel electrophoresis, targetband size between500-700bp. Trichophyton rubrum and trichophyton violaceum sharedprobe Trirv, microsporum canis and microsporum ferrugineum shared probe Miccf,trichophyton tonsurans and trichophyton schoenleinii shared probe Trits, other strainshave their own species-specific probe.ConclusionFungal DNA was simplely and rapidly extracted by the Biospin fungal genomicDNA extraction kit. The Array Designer4software has met the demand of design specific oligonucleotide probes in ITS1or ITS2. ObjectiveThe Nano-gold gene chips were prepared and applied to identify fungal strains（standard strains, superficial fungi and negative control strains）.MethodsThe aldehyde modiflied slide fixed detection probe, positive control probe andnegative control probe. The Nano-gold labeled streptavidin incubated after the PCRproduct was hybridized with the oligonucleotide microarray, then formated thebiotin-streptavidin-Nano-gold amplification system. And finally the signal was amplifiedby silver enhancement staining.ResultsThe5’end amino-modified fungus-specific oligonucleotide probes were reacted onaldehyde modiflied slide then fix on the slide firmly. The biotin-streptavidin-Nano-goldamplification system combined with silver enhancement staining, the detection resultscould be easily recognized by naked eyes. Fungal standard strains, common superficialfungi strains, and negative control strains were tested by the above method. The resultsshowed that17standard strains，32dermatophytes,33yeast corresponded with theirown specific probe and positive control probe, did not corresponded with negative control probe and the other probe.2negative control strains only corresponded with positivecontrol probe.The results were consistent with that by the traditional diagnostic methodssuch as microscopy and culture.ConclusionThe Gold Nanoparticles probe-based DNA microarray was proved to be a quick andaccurate identification system for pathogenic fungi species.