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A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five Aspergillus Species
Maria Tokamani1, Eleftheria Figgou1, Lito Papamichail1
1Department of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greece.
Abstract:
Aspergillus mold is a ubiquitously found, airborne pathogen that can cause a variety of diseases from mild to life-threatening in severity. Limitations in diagnostic methods combined with anti-fungal resistance render Aspergillus a global emerging pathogen. In industry, Aspergilli produce toxins, such as aflatoxins, which can cause food spoilage and pose public health risk issues. Here, we report a multiplex qPCR method for the detection and identification of the five most common pathogenic Aspergillus species, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans. Our approach exploits species-specific nucleotide polymorphisms within their ITS genomic regions. This novel assay combines multiplex single-color real time qPCR and melting curve analysis and provides a straight-forward, rapid, and cost-effective detection method that can identify five Aspergillus species simultaneously in a single reaction using only six unlabeled primers. Due to their unique fragment lengths, the resulting amplicons are directly linked to certain Aspergillus species like fingerprints, following either electrophoresis or melting curve analysis. Our method is characterized by high analytical sensitivity and specificity, so it may serve as a useful and inexpensive tool for Aspergillus diagnostic applications both in health care and the food industry.
Insights
A new multiplex qPCR method rapidly detects five common pathogenic Aspergillus species. This cost-effective assay uses species-specific DNA regions for accurate identification in healthcare and food industries.
Area of Science:
- Mycology
- Molecular Biology
- Infectious Diseases
Background:
- Aspergillus mold is an airborne pathogen causing diverse diseases and producing harmful toxins like aflatoxins.
- Limitations in current diagnostic methods and antifungal resistance highlight the need for improved Aspergillus detection.
- Aspergillus species pose significant risks in both healthcare settings and the food industry due to spoilage and public health concerns.
Purpose of the Study:
- To develop a novel multiplex quantitative polymerase chain reaction (qPCR) assay.
- To enable simultaneous detection and identification of five common pathogenic Aspergillus species.
- To provide a rapid, cost-effective, and accurate diagnostic tool for Aspergillus.
Main Methods:
- Exploited species-specific nucleotide polymorphisms within the Internal Transcribed Spacer (ITS) genomic regions of Aspergillus.
- Developed a multiplex single-color real-time qPCR assay combined with melting curve analysis.
- Utilized six unlabeled primers for simultaneous amplification and identification of target species based on unique amplicon fragment lengths.
Main Results:
- Successfully detected and identified five common pathogenic Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, A. nidulans) simultaneously in a single reaction.
- Achieved high analytical sensitivity and specificity.
- Demonstrated that amplicon fragment lengths act as species-specific fingerprints detectable via electrophoresis or melting curve analysis.
Conclusions:
- The developed multiplex qPCR assay is a straightforward, rapid, and cost-effective method for Aspergillus diagnostics.
- This assay can identify five key pathogenic Aspergillus species simultaneously.
- The method offers a valuable tool for Aspergillus detection in both clinical healthcare and food industry applications.
