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Updated: Jun 27, 2026
Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB
Published on: August 6, 2011
Development of PCR-Multiplex Assays for Identification of the Herpotrichiellaceae Family and Agents Causing
Gabriel S M Sousa1,2, Rodrigo S De Oliveira2, Alex B Souza2
1Programa de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém 66075-750, Brazil.
Abstract:
The Herpotrichiellaceae family is an important group of dematiaceous filamentous fungi, associated with a variety of pathogenic fungal species causing chromoblastomycosis (CBM) and phaeohyphomycosis (PHM), both with polymorphic clinical manifestations and worldwide incidence. Currently, the identification of this family and determination of the causative agent is challenging due to the subjectivity of morphological identification methods, necessitating the use of molecular techniques to complement diagnosis. In this context, genetic sequencing of the Internal Transcribed Spacer (ITS) has become the norm due to a lack of alternative molecular tools for identifying these agents. Therefore, this study aimed to develop PCR-Multiplex methodologies to address this gap. Sequences from the ITS and Large Subunit (LSU) of ribosomal DNA were used, and after manual curation and in vitro analyses, primers were synthesized for the identification of the targets. The primers were optimized and validated in vitro, resulting in two PCR-Multiplex methodologies: one for identifying the Herpotrichiellaceae family and the bantiana clade, and another for determining the species Fonsecaea pedrosoi and Fonsecaea monophora. Ultimately, the assays developed in this study aim to complement other identification approaches for these agents, reducing the need for sequencing, improving the management of these infections, and enhancing the accuracy of epidemiological information.
Insights
New PCR-Multiplex methods can now quickly identify fungal agents causing chromoblastomycosis and phaeohyphomycosis. These molecular tools improve diagnosis and management of these challenging infections.
Area of Science:
- Medical Mycology
- Molecular Biology
- Infectious Diseases
Background:
- Herpotrichiellaceae fungi cause chromoblastomycosis and phaeohyphomycosis globally.
- Current identification relies on subjective morphology and ITS sequencing.
- A need exists for more efficient diagnostic molecular tools.
Purpose of the Study:
- Develop novel PCR-Multiplex assays for Herpotrichiellaceae identification.
- Create methods to differentiate key pathogenic species.
- Provide alternatives to traditional sequencing methods.
Main Methods:
- Utilized Internal Transcribed Spacer (ITS) and Large Subunit (LSU) ribosomal DNA sequences.
- Designed and synthesized specific primers for PCR-Multiplex assays.
- Validated assays in vitro for accuracy and specificity.
Main Results:
- Developed two PCR-Multiplex methodologies.
- One assay identifies the Herpotrichiellaceae family and bantiana clade.
- The second assay differentiates Fonsecaea pedrosoi and Fonsecaea monophora.
Conclusions:
- The developed PCR-Multiplex assays offer rapid and accurate identification of key fungal pathogens.
- These methods reduce reliance on sequencing, aiding clinical and epidemiological efforts.
- Improved diagnostics can lead to better patient management and infection control.
