Rapid identification of clinically important Aspergillus species by Polymerase Chain Reaction: Restriction Fragment Length Polymorphism
Majid ZARRIN, Farzaneh GANJ, Maryam ERFANINEJAD
Department of Medical Mycology, Medical School, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Introduction: Making a diagnosis of aspergillosis is complex and requires a combination of clinical, radiological and serological findings for interpretation. Current conventional methods are time consuming with low sensitivity. Management of aspergillosis requires rapid and accurate identification of Aspergillus (A.) species in order for treatment to be started as soon as possible. The objective of this pilot study was to evaluate a novel rapid method for discrimination of the most clinically relevant pathogenic A. species. Materials and Method: A total of 33 A. isolates including A. fumigatus, A. niger, A. flavus and A. terreus were studied. DNA extraction from fungal strains was performed using the phenol/chloroform method. Primers internal transcribed spacer 1(ITS1) and internal transcribed spacer 2(ITS2) were used in this study to generate polymerase chain reaction (PCR) product of about 600 bp for each A. species. The PCR amplicons from each species were incubated with 10 unit of Bacillus globigii I (Bg/I) enzyme and subsequently electrophoresed in 2.5% gel agarose. Restriction enzyme patterns of the A. species sequences were predicted for restriction endonucleases. Result: The ITS1 and ITS4 primers were able to amplify the ITS1–5.8S rDNA–ITS2 region of the tested isolates, providing a single PCR product of about 600 bp for each A. species. The digested products with Bg/I restriction enzyme produced smaller varying fragment sizes with different restriction enzyme patterns on gel electrophoresis, allowing for the discrimination of the four most clinically relevant pathogenic A. species, within a day. Repeated experiment carried out for the 33 A. isolates yielded similar results. Conclusion: Our novel method is the first to describe using ITS region nucleotide sequences of common A. species and a Bg/I enzyme digested PCR-restriction fragment length polymorphism profile can allow for rapid discrimination of the most clinically important A. species.
Keywords: polymerase chain reaction-restriction fragment length polymorphism, Aspergillus species, internal transcribed spacer region, diagnosis
Correspondence author: Majid ZARRIN, Department of Medical Mycology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
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Brunei Int Med J. 2016; 12 (4): 140-143