Partial Alpha-Tubulin Gene Amplification by Nested PCR in Fresh Biological Samples and Formalin-Fixed,
Igor Massahiro de Souza Suguiura1, Eiko Nakagawa Itano2, Mario Augusto Ono3
1Laboratory of Animal Immunology, Department of Immunology, Parasitology and General Pathology, State University of Londrina, Londrina, Brazil. igormassahiro@hotmail.com.
Mycopathologia
|June 8, 2025
Summary
A new nested PCR method accurately identifies five Paracoccidioides species from biological samples. This diagnostic tool aids in distinguishing Paracoccidioidomycosis, a significant fungal infection.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Paracoccidioidomycosis is a prevalent deep mycosis in Latin America.
- The causative agents of Paracoccidioidomycosis have been reclassified into five distinct species.
- Accurate and rapid diagnostic methods are crucial for effective treatment and control.
Purpose of the Study:
- To develop and validate a novel nested PCR method for the diagnosis and species differentiation of Paracoccidioides species.
- To assess the efficacy of the method in detecting Paracoccidioides DNA directly from biological samples.
- To evaluate the compatibility of the generated amplicons with downstream analysis techniques.
Main Methods:
- Nested PCR targeting the partial alpha-tubulin gene of Paracoccidioides species.
- Testing the method's specificity against other fungal pathogens.
- Application of the nested PCR on fresh and formalin-fixed paraffin-embedded (FFPE) animal tissues.
- Downstream analysis including Restriction Fragment Length Polymorphism (RFLP) and DNA sequencing.
Main Results:
- The nested PCR successfully amplified DNA from all five cultivable Paracoccidioides species.
- The method demonstrated high specificity, not amplifying DNA from other tested fungal species.
- Detection of Paracoccidioides spp. was achieved in both fresh and FFPE tissues.
- Formalin fixation introduced base changes in FFPE-derived amplicons, interfering with RFLP analysis but not preventing sequencing.
Conclusions:
- The developed nested PCR is a sensitive and specific tool for diagnosing Paracoccidioidomycosis and differentiating between its causative species directly from clinical samples.
- The method shows promise for application with both fresh and FFPE tissues, although FFPE samples require careful consideration for downstream enzymatic analyses.
- Further optimization for FFPE samples may enhance its utility in retrospective studies and routine diagnostics.
