DIFFERENTIATION OF ASPERGILLUS SPECIES USING AUTOMATED REP-PCR DNA FINGERPRINTING

Houng J, Webb R, Manry J, Raza S, Bittner T, Reece K, Healy M

Author address: 

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Abstract: 

Background: Aspergillus has been increasingly recognized as an invasive pathogen, with about 20 species reported as causative agents of opportunistic infections in humans. Among these, Aspergillus fumigatus is the most commonly isolated species, followed by Aspergillus flavus and Aspergillus niger. This fungus causes allergic diseases in asthmatics and patients suffering from cystic fibrosis, as well as, sinusitis, cerebral aspergillosis, meningitis, endocarditis, myocarditis, and pulmonary aspergillosis. Traditional microscopic morphology provides identification only at the genus/species level and may take several days to perform. Other molecular methods may be less automated (PFGE), tedious (RFLP), and are prone to artifacts (RAPD). A commercial assay, automating rep-PCR, has shown promise for rapid fungal identification and strain classification. A microfluidics device separates the amplified products and the results are then analyzed by the DiversiLab System Software, which also provides a report. This study explores the performance of the DiversiLab System as an alternative molecular genotyping tool for Aspergillus spp. Materials and Methods: 50 clinical and non-clinical isolates of previously characterized Aspergillus species (including A. fumigatus, A. flavus, A. terrus, A. niger) were analyzed. Approximately 50ng of DNA was amplified using the DiversiLab DNA Fingerprinting Kit. The amplified product (1 ul) was analyzed using the Caliper 1000 Analyzer and the data was processed using the system software. Results: The results show differentiation of Aspergillus species concordant to previous characterization by traditional methods. Significant genetic diversity was appreciated within samples of the same genus/species. Reproducibility was very high (100 %) and profiles were easy to interpret. This method gave results for all the samples, which were processed and analyzed in 1 working day by a single technician. Conclusions: The DiversiLab System could be a time-efficient, effective and easy to use, novel genotyping tool for the identification and strain relatedness of fungi. The system could also be applicable in determining source of fungal infections.
2003

abstract No: 

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Full conference title: 

The 15 th Congress of the International Society for Human and Animal Mycology
    • ISHAM 15th (2003)