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Aspergillus collagen-like genes (acl): identification, sequence polymorphism, and assessment for PCR-based pathogen
Kiril Tuntevski1, Brandon C Durney, Anna K Snyder
1Department of Microbiology, Immunology, and Cell Biology.
Abstract:
The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.
Insights
This study introduces a new PCR method targeting Aspergillus collagen-like (acl) genes for rapid species identification. This approach aids in diagnosing Aspergillus infections, especially in vulnerable patients.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Genetics
Background:
- The genus Aspergillus poses significant public health risks due to its environmental ubiquity, allergenicity, and susceptibility in patients with cystic fibrosis, asthma, and compromised immunity.
- Current diagnostic methods for Aspergillus colonization and infection, such as morphological characterization and serological assays, are time-consuming and limited in scope.
- Collagen-like genes offer potential for species-specific identification due to conserved and polymorphic regions.
Purpose of the Study:
- To assess the conserved regions of Aspergillus collagen-like (acl) genes for PCR-based species discrimination.
- To explore the application of PCR amplicon size-based discrimination for five key Aspergillus species: A. fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus.
- To investigate the utility of acl genes in identifying Aspergillus species in clinical samples and to enhance discrimination using capillary electrophoresis.
Main Methods:
- Analysis of conserved regions within Aspergillus collagen-like (acl) genes.
- Development and application of PCR assays for amplicon size-based discrimination.
- Examination of genetic polymorphism and phylogenetic analysis of the aclF1 gene.
- Testing the PCR assay on clinical samples (sputum, bronchoalveolar fluid).
- Utilizing capillary electrophoresis on nanogels for improved species discrimination.
Main Results:
- Aspergillus acl genes were successfully used as PCR targets to differentiate between clinically relevant Aspergillus species.
- PCR amplicon size-based discrimination was effective for the five common etiologic species.
- Genetic polymorphism and phylogenetic analysis of aclF1 provided further insights.
- The PCR-based assay showed applicability in identifying Aspergillus species from clinical cultures.
- Capillary electrophoresis enhanced the discrimination capabilities between Aspergillus species.
Conclusions:
- Aspergillus acl genes are viable PCR targets for discriminating between clinically significant Aspergillus species.
- This molecular approach offers a promising alternative to traditional diagnostic methods.
- Future research will focus on optimizing PCR and microfluidic applications for sensitive and specific detection of Aspergillus colonization and invasive aspergillosis in immunocompromised individuals.
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