Detection of Aspergilus DNA, by a Nested PCR assay and molecula study of Aspergillus fumigatus, by PCR amplification of IGS Region in BMT patients

F. Teifoori1, S. Rodbar Mohammadi1, Z. Sharifi2 and H. Ghaffari3

Author address: 

1Tarbiat Modares University, Iran, 2Iran Blood transfusion Organization, Iran and 3Bone marrow transplantation unit, shariati Hospital, Iran

Abstract: 

ntroduction and objectives: The incidence of invasive aspergillosis has undeniably risen over the past two decades. Diagnosing of IA in immunocompromised patients,has clinical problem. Molecular tools are gaining importance in detection of Aspergillus DNA in Blood. In this survey our goal would be to investigate the presence of Aspergillus fumigatus in BMT patients using by PCR method. For detection of electrophoretic patterns of Aspergillus fumigatus, IGS gene was used. Materials and Methods: A total of 30 BMT patients blood samples were collected during 3 years. A. Fumigatus DNA was extracted from blood samples by Bahar Afshan kit. Then Aspergillus fumigatus strain: 5009 PTCC cultured on potato dextrose agar at 37 C for 5 days and mycelium was harvested. DNA of mycelium extracted using by phenol chlorohorm method and PCR was performed with 50 ml primer (1st step: AFU7S/AFU7AS) and (2nd step: AFU5S/AFU5AS) with annealing temperature: 60 C, That specifically amplifies a region of the 18S rRNA gene that is higly conserved in Aspergillus species. Also, IGS PCR was done with two primers (IGSL/IGSR), that designed to amplify the intergenic spacer regions between ribosomal DNA transcription units with annealing temperature: 45 C. The PCR products were visualized by 1% agarose gel electrophoresis, stained with ethidium loromide. Fusarium oxysporum, Aspergillus flavus and water were used as negative controls. The 50 bp and 100 bp ladder was used as marker. Results: Results showed that with using Nested PCR assay 236 bp 420 bp fragments was separated from Aspergillus fumigatus,so these specific primers can differentiated Aspergillus fumigatus among immunocompromised and BMT patients (n = 30), the PCR results of six patients were correctly positive. The results of IGS PCR revealed That the two primers, (IGSL/IGSR) differentiated 30 BMT patients blood samples of aspergillosis cases with different source and molecular pattern. Conclusions: Invasive aspergilosis in neutropenia and recipient bone marrow patients is one of the life-threatening diseases. In vitro identification methods such as phenotypic methods have low sensitivity and are time consuming. So that today, one of the most accurate techniques are based on molecular methods in our study Nested PCR with specific primers for Aspergillus fumigatus has shown to be sensitive diagnostic tool for detection of infectious pathogens in at risk patients. Also the PCR amplification of IGS region of A. fumigatus is a method for subspecific typing of this organism.
2011

abstract No: 

P075

Full conference title: 

Trends in Medical Mycology, 5th
    • TIMM 5th (2013)