An improved rapid quantitative detection and identification method for a wide range of fungi

Abstract

To develop a rapid and quantitative diagnostic technique for the detection and identification of a wide range of fungi, an improved molecular method based on real-time PCR and the analysis of its products that targets the internal transcribed spacer (ITS) 2 region was established. The real-time PCR could quantitatively and specifically detect the ITS2 region from all 24 tested pathogenic fungal species at between 10 1 and 10 7 copies per test without amplification of bacterial or human DNA. The sequences of the primer-binding sites are conserved in the registered sequences of 34 other pathogenic fungal species, suggesting that the PCR would also detect these species. The hyperpolymorphic nature of the ITS2 region between fungal species in terms of length and nucleotide sequence provided valuable information for the determination of species. By labelling the 59 end of the reverse primer with NED fluorescent dye, the fragment lengths of the real-time PCR products and their 39-terminal fragments, derived using restriction enzyme ScrFI digestion, were easily evaluated by capillary electrophoresis. Using this analysis, the number and species of fungi present in samples could be estimated. Moreover, sequence analysis of the real-time PCR products could accurately determine species in samples containing a single species. This diagnostic technique can estimate a wide range of fungi from various clinical samples within 1 day and accurately identify them in 2 days. Quantitative results for fungal titre in samples can also provide useful information for understanding the progression of disease and the efficacy of antifungal chemotherapy.

Review article: fungal microbiota and digestive diseases

“…Fungal phylogeny can be reconstructed from the molecular level, such as in the sequences of ribosomal RNA and other highly conserved genes . At present, the DNA sequencing based on the fungal 18S rDNA and internal transcribed spacer (ITS) has been widely applied in fungal microbiota . 18S rDNA sequence has conserved and variable regions, where conserved areas reflect phylogenetic relationships among species, highly variable regions reflect the differences between species.…”

“…42 At present, the DNA sequencing based on the fungal 18S rDNA and internal transcribed spacer (ITS) has been widely applied in fungal microbiota. 43,44 18S rDNA sequence has conserved and variable regions, where conserved areas reflect phylogenetic relationships among species, highly variable regions reflect the differences between species. 18S rDNA sequence can be applied to classify fungi at the species level and above.…”

Childhood Tinea Incognito Caused by Trichophyton mentagrophytes var. interdigitale Mimicking Pustular Psoriasis

“…Another set of the genotyping for internal transcribed spacer (ITS2) region hit the sequence homology consistent with T. mentagrophytes var. interdigitale and T. rubrum (1) . From these results, she was diagnosed as tinea incognito (TI) (2), which was caused by T. mentagrophytes var.…”

Antifungal agent production from a new marine Bacillus pumilusSMH101

“…In recent years, the incidence of invasive opportunistic fungal infections has been increasing due to increases in the number of immune-compromised patients (Soeta et al, 2009). Infections due to Candida species in immunecompromised patients are the most common; however, Aspergillus and other pathogenic fungi are also emerging as a threat to public health (Richardson, 2005).…”