Abstract
Aspergillus fumigatus remains the most frequent cause of invasive aspergillosis; however, other species, including Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Aspergillus nidulans and Aspergillus ustus have been reported to cause human infection (Henry et al., 2000). Rapid and accurate identification of Aspergillus species is necessary for successful clinical management of infection and for epidemiological purposes. Identification of Aspergillus species based on morphological methods requires adequate growth time for evaluation of colony characteristics and microscopic features. A culture time of 5 days or more is generally required for the development of anamorphic forms of Aspergillus. Failure to form conidia on ordinary culture media may require colonies to be further subcultured on specialized media to induce spore formation (Henry et al., 2000; Hinrikson et al., 2005). In addition, morphology tests are usually labour intensive and need expert mycology personnel. Due to these limitations, various molecular approaches have been used for the identification of Aspergillus species isolated from clinical samples, including PCR amplification of targets followed by either fragment length analysis or DNA probe hybridization or sequence analysis (Hinrikson et al., 2005). The aim of this study was to compare the internal transcribed spacer 1 (ITS1)–ITS2 nucleotide sequences of common Aspergillus species and design a PCR-RFLP profile for differentiation of the most medically important Aspergillus species.
Standard strains were provided by Teikyo University Institute of Medical Mycology, Tokyo, Japan. The ITS region of the strains was sequenced using the universal fungal primers ITS1 and ITS4 for reconfirmation of the species, and for using in enzyme selection for RFLP analysis. In addition, 33 clinical and environmental isolates were also used (Table 1). The isolates were phenotypically identified according to the macroscopic and microscopic morphology of their colonies on Czapek–Dox agar (Difco). All moulds were also cultured on Sabauroud's dextrose agar and incubated at 30 °C for 2 days. Genomic DNA was extracted using single-tube rapid glass-bead disruption (Yamada et al., 2002). Total DNA in the supernatant was precipitated with 2-propanol, resuspended in 100 µl deionized water, and preserved at –20 °C until use. Glass-bead preparation has been used repeatedly for DNA extraction from yeasts (Mirhendi et al., 2006; Yamada et al., 2002). We found that the method is easy to perform and inexpensive tool for rapid extraction of DNA with good recovery.