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Aspergillus section Fumigati typing by PCR-restriction fragment polymorphism
Janet F Staab1, S Arunmozhi Balajee, Kieren A Marr
1Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Abstract:
Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from A. fumigatus. The sequence data show that the gene encoding beta-tubulin, benA, has high interspecies variability at intronic regions but is conserved among isolates of the same species. These data were used to develop a PCR-restriction fragment length polymorphism (PCR-RFLP) method that rapidly and accurately distinguishes A. fumigatus, A. lentulus, and N. udagawae, three major species within the section Fumigati that have previously been implicated in disease. Digestion of the benA amplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benA sequences of Aspergillus spp. deposited in the GenBank database. PCR-RFLP of benA is a simple method for the identification of clinically important, similar morphotypes of Aspergillus spp. within the section Fumigati.
Insights
Distinguishing Aspergillus Fumigati species is crucial for antifungal susceptibility. A new PCR-RFLP method using the benA gene accurately identifies Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae.
Area of Science:
- Medical Mycology
- Molecular Biology
- Clinical Microbiology
Background:
- Several Aspergillus section Fumigati species, including Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae, are morphologically similar.
- Accurate identification is clinically important due to varying antifungal susceptibilities among these species.
- Previous work established A. lentulus as distinct from A. fumigatus using multilocus sequence typing.
Purpose of the Study:
- To develop a rapid and accurate molecular method for distinguishing key Aspergillus section Fumigati species.
- To differentiate between Aspergillus fumigatus, A. lentulus, and N. udagawae based on genetic markers.
Main Methods:
- Utilized multilocus sequence typing data, specifically focusing on the beta-tubulin (benA) gene.
- Developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay targeting polymorphic intronic regions of the benA gene.
- Validated the PCR-RFLP method using clinical isolates and in silico analysis of Aspergillus spp. sequences from GenBank.
Main Results:
- The benA gene exhibits high interspecies variability in intronic regions, conserved within species.
- Digestion of the benA amplicon with BccI restriction enzyme produced unique banding patterns for each target species.
- The PCR-RFLP method demonstrated high accuracy in distinguishing A. fumigatus, A. lentulus, and N. udagawae.
Conclusions:
- PCR-RFLP of the benA gene provides a simple, rapid, and accurate method for identifying clinically relevant, morphologically similar Aspergillus species.
- This molecular technique aids in the correct identification of disease-causing Aspergillus spp. within the Fumigati section.
- Facilitates appropriate antifungal treatment selection by enabling precise species identification.
