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PCR and real-time PCR assays to detect fungi of Alternaria alternata species
Milena Kordalewska1, Anna Brillowska-Dąbrowska1, Tomasz Jagielski2
1Department of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdańsk University of Technology, Gdańsk, Poland.
Abstract:
Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens.
Insights
New PCR and real-time PCR assays enable rapid and accurate detection of Alternaria alternata, a fungus increasingly causing infections in immunocompromised individuals. This advancement aids in timely diagnosis of phaeohyphomycosis.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Fungi in the Alternaria genus are commonly linked to allergies.
- Emerging infections (phaeohyphomycoses) caused by Alternaria species, particularly Alternaria alternata, are increasingly observed in immunocompromised patients.
- Current diagnostic methods relying on morphology are slow and often inconclusive, delaying diagnosis.
Purpose of the Study:
- To develop and validate simple, reliable Polymerase Chain Reaction (PCR) and real-time PCR assays for the specific detection of Alternaria alternata.
- To improve the speed and accuracy of diagnosing Alternaria alternata infections.
Main Methods:
- Design of Alternaria alternata-specific primers based on β-tubulin gene sequence alignment.
- DNA extraction from fungal isolates using a two-step procedure.
- Application of PCR and real-time PCR assays, followed by electrophoresis or melting temperature analysis for detection.
Main Results:
- The developed PCR and real-time PCR assays demonstrated high specificity, accurately identifying all tested Alternaria alternata isolates.
- No cross-reactivity was observed with other fungi (molds, dermatophytes, yeast-like fungi) or human DNA.
- The assays provide fast and unambiguous identification of Alternaria alternata.
Conclusions:
- The developed molecular assays offer a significant improvement over traditional methods for identifying Alternaria alternata.
- These assays facilitate rapid and precise diagnosis of phaeohyphomycosis caused by Alternaria alternata, crucial for effective patient management.
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