Single strand conformation polymorphism analysis of PCR-amplified rDNA to differentiate medically important Aspergillus species

ABSTRACT

Aspergillus species are associated with allergic bronchopulmonary disease, mycotic keratitis, otomycosis, nasal sinusitis and invasive infection. In this study, we developed a PCR-Single Strand Conformational Polymorphism method to identify the most common Aspergillus species and we showed some advantages of this method comparing a PCR-Restriction Fragment Length Polymorphism with our designed restriction enzyme. We selected ITS2, as a short fragment within the rDNA region [length size 330 bp] to be amplified as small size PCR product. We mixed 5 ml of the PCR product with an equal volume of loading buffer and followed by incubation for 5 min at 95°C and quenching in an ice bath. The mixture was applied to a 6%-12% Gradient Poly acryl amide gel to run in a vertical electrophoresis, then gel was stained with ethidium bromide and silver nitrate which followed by an ethidium bromide staining. Our results of restriction digestion showed a fine identification of 7 tested Aspergillus species during 5-6 hours after an overnight mycelial growth. As our results some of tested Aspergillus species A. nidulans, A.fisheri, A. quadricincta, [A. fumigatus and A. niger] as a group and [A. flavus, A. tereus and A. ochraceus] as another group, can be discriminated. Moreover SSCP analysis enabled us to identify above Aspergillus species within 8-12 h after an over night growth without using an expensive restriction enzyme. It is concluded that Single Strand Conformational Polymorphism is a simple and rapid method for identification of some medically important Aspergillus