Methods: DNA extraction was performed from cultures of medically important fungi, including C.albicans (ATCC 90029), C.neoformans (ATCC 34875), A.fumigatus and Fusarium spp.. To model simple fungal community, the mixtures of fungal-specific DNA (C.albicans:C.neoformans:A.fumigatus:Fusarium spp.) were prepared at ratios of 1:1:1:1, 0.1:1:1:1, 0.01:1:1:1, 0.001: 1:1:1 respectively. To examine the abundance and diversity of fungal-specific in DNA mixtures, the quantitative SYBR PCR (qPCR) and terminal fragment length polymorphisms (TRFLP) were performed. For qPCR, DNA was amplified using species-specific primers and universal primers targeting fungal internal transcribed spacer (ITS) region, and the ratios of each fungi:total fungi were estimated. For TRFLP, DNA mixtures were amplified using the fluorescent-labeled forward-primer and the unlabeled reverse-primer targeting the ITS region. PCR products were digested with MspI or HaeIII endonuclease enzymes followed by fragment purification. DNA fragments were analysed using the Applied Biosystems 3130 series Genetic Analyzers. Fungal-specific peaks were estimated and TRFLP profiles were produced using the GeneMapper4.0.
Results: The qPCR could detect 2-10 copies of targets whereas the detection by TRFLP method required at least 10-100 copies. Fungal detection by both qPCR and TRFLP method were highly specific to each fungus in all DNA mixtures and specific products were shown in DNA gels. Both MspI- or HaeIII-digestion provided specific fragments to each fungus in all DNA mixtures. In contrast to qPCR method, the abundance of species-specific TRF fragments in fungal community could not be accurately estimated.
Conclusions: TRFLP with MspI- or HaeIII-digestion may be used to determine the oral fungal community when the target copy number exceed 100. In addition to TRFLP, qPCR method is required for accurately estimate abundance of the fungi in the community.