Development of RFLP-PCR method for the identification of medically important Aspergillus species using single restriction enzyme MwoI

In this study we attempted to modify the PCR-RFLP method using restriction enzyme MwoI for the
identification of medically important Aspergillus species. Our subjects included nine standard
Aspergillus species and 205 Aspergillus isolates of approved hospital acquired infections and hospital
indoor sources. First of all, Aspergillus isolates were identified in the level of species by using
morphologic method. A twenty four hours culture was performed for each isolates to harvest
Aspergillus mycelia and then genomic DNA was extracted using Phenol-Chloroform method.
PCR-RFLP using single restriction enzyme MwoI was performed in ITS regions of rDNA gene. The
electrophoresis data were analyzed and compared with those of morphologic identifications. Total of
205 Aspergillus isolates included 153 (75%) environmental and 52 (25%) clinical isolates. A. flavus
was the most frequently isolate in our study (55%), followed by A. niger 65(31.7%), A. fumigatus
18(8.7%), A. nidulans and A. parasiticus 2(1% each). MwoI enabled us to discriminate eight medically
important Aspergillus species including A. fumigatus, A. niger, A. flavus as the most common
isolated species. PCR-RFLP method using the restriction enzyme MwoI is a rapid and reliable test for
identification of at least the most medically important Aspergillus species.