Morphological identification of the
Translation elongation factor (TEF) 1-α consistently presents as a single-copy gene in the
Fifty strains of
Thick spore suspension (1 ml) was inoculated in Ehrlenmeyer flasks containing yeast extract peptone dextrose medium and incubated on an incubator shaker at 200 rpm under agitation for 72 h at 25°C for mycelia growth. The harvested mycelia were washed with 0.5 M EDTA and sterile dH2O. The mycelia were ground into a fine powder using liquid nitrogen and a mortar and pestle.
Approximately 100 mg powdered mycelium was transferred into a 1.5-ml tube containing 400 µl lysis buffer (100 mM Tris-HCl, pH 8.0, 30 mM EDTA, pH 8.0, 5% SDS w/v). After microtubes were boiled at 100°C for 20 min, 3 M acetate potassium (150 µl) was added to each tube. The suspension was maintained at −20°C for 10 min and centrifuged at 14,000 × g in 4°C for 10 min. The supernatant was carefully transferred to a fresh 1.5-ml Eppendorf tube and 250 µl phenol:chloroform:isoamyl alcohol (25:24:1, v/v) was added. The microtube was vortexed briefly and centrifuged at 4°C at 14,000 × g for 10 min. After transferring the supernatant to a 1.5-ml microtube, 250 µl chloroform:isoamyl alcohol was added. The tubes were briefly vortexed and centrifuged at 4°C at 14,000 × g for 10 min. The supernatant was transferred to a fresh microtube, an equal volume of ice-cold 2-propanol was added, maintained at −20°C for 10 min and centrifuged at 14,000 × g for 10 min. The upper aqueous phase was discarded and the pellet was washed with 70% ethanol (300 µl). The ethanol was discarded and the DNA pellets were air dried and resuspended in 50 µl dH2O.
The primer sets, Fu3f (5′-GGTATCGACAAGCGAACCAT-3′) and Fu3r (5′-TAGTAGCGGGGAGTCTCGAA-3′) was used to amplify an ~420-bp DNA fragment of the TEF-1α gene (
An initial denaturation step for 5 min at 94°C was followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min and extension at 68°C for 2 min. The amplified PCR product (5 µl) was electrophoresed on 1% agarose gel in TAE buffer at 100 V for 1 h and stained with ethidium bromide. The PCR amplification of TEF-1α gene resulted in an ~420-bp fragment.
Digestion with one restriction enzyme was not sufficient to discriminate the 420-bp DNA fragment of the TEF-1α gene in the
The PCR amplification of TEF-1α gene with Fu3f and Fu3r primers produced a unique band of ~420 bp for all tested
Double digestion of the fragment with restriction enzymes,
The restriction patterns of one clinical and six environmental
Identification of filamentous fungi at the species level using classical techniques, such as morphological methods, is difficult and time-consuming. Novel rapid techniques are required in order to verify the
The majority of molecular techniques are PCR-based, where the primers are typically directed to conserved regions of the ribosomal DNA gene, particularly towards the internal transcribed spacer (ITS) regions. With regard to
Guevara-Suarez
In the current study, molecular identification was performed using the TEF-1α gene and RFLP, and it was possible to discriminate between all five clinically important
The Primer set, TEF-Fu3 resulted in an ~420-bp product for five of the
The present study was supported by the Health Research Institute, Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences (Ahvaz, Iran) (grant no. 92118).
Agarose gel electrophoresis of transcription elongation factor-1α gene products (420 bp) of the
Agarose gel electrophoresis of transcription elongation factor-1α gene products (420 bp) of the
Agarose gel electrophoresis of transcription elongation factor-1α gene products (420 bp) of the
Restriction fragment size (bp) of the
| TEF-1α fragment prior to digestion (bp) | ||
|---|---|---|
| 420 | 45,62,103,170 | |
| 420 | 6,30,56,47,55,186 | |
| 420 | 25,168,187 | |
| 420 | 27,62,99,192 | |
| 420 | 308,110 |
TEF, transcription elongation factor.