A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five <i>Aspergillus</i> Species
<i>Aspergillus</i> 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 <i>Aspergillus</i> a global emerging pathogen. In i...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-08-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/9/8/842 |
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| author | Maria Tokamani Eleftheria Figgou Lito Papamichail Eleni Sakka Athanasios Toros Anastasia Bouchorikou Antonis Giannakakis Efthymia Iliana Matthaiou Raphael Sandaltzopoulos |
| author_facet | Maria Tokamani Eleftheria Figgou Lito Papamichail Eleni Sakka Athanasios Toros Anastasia Bouchorikou Antonis Giannakakis Efthymia Iliana Matthaiou Raphael Sandaltzopoulos |
| author_sort | Maria Tokamani |
| collection | DOAJ |
| description | <i>Aspergillus</i> 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 <i>Aspergillus</i> a global emerging pathogen. In industry, <i>Aspergilli</i> 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 <i>Aspergillus</i> species, <i>Aspergillus fumigatus</i>, <i>Aspergillus flavus</i>, <i>Aspergillus niger</i>, <i>Aspergillus terreus</i>, and <i>Aspergillus nidulans.</i> 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 <i>Aspergillus</i> 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 <i>Aspergillus</i> 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 <i>Aspergillus</i> diagnostic applications both in health care and the food industry. |
| first_indexed | 2024-03-10T23:49:37Z |
| format | Article |
| id | doaj.art-8bd9694a2ccf41e184a6cd8ee3325ce3 |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-10T23:49:37Z |
| publishDate | 2023-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-8bd9694a2ccf41e184a6cd8ee3325ce32023-11-19T01:47:50ZengMDPI AGJournal of Fungi2309-608X2023-08-019884210.3390/jof9080842A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five <i>Aspergillus</i> SpeciesMaria Tokamani0Eleftheria Figgou1Lito Papamichail2Eleni Sakka3Athanasios Toros4Anastasia Bouchorikou5Antonis Giannakakis6Efthymia Iliana Matthaiou7Raphael Sandaltzopoulos8Department of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, GreeceDepartment of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, GreeceDepartment of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, GreeceDepartment of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, GreeceDepartment of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, GreeceDepartment of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, GreeceDepartment of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, GreeceDepartment of Medicine, Division of Pulmonary Allergy and Critical Care Medicine, School of Medicine, Stanford University, Stanford, CA 94305, USADepartment of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greece<i>Aspergillus</i> 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 <i>Aspergillus</i> a global emerging pathogen. In industry, <i>Aspergilli</i> 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 <i>Aspergillus</i> species, <i>Aspergillus fumigatus</i>, <i>Aspergillus flavus</i>, <i>Aspergillus niger</i>, <i>Aspergillus terreus</i>, and <i>Aspergillus nidulans.</i> 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 <i>Aspergillus</i> 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 <i>Aspergillus</i> 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 <i>Aspergillus</i> diagnostic applications both in health care and the food industry.https://www.mdpi.com/2309-608X/9/8/842<i>Aspergillus fumigatus</i><i>Aspergillus flavus</i><i>Aspergillus niger</i><i>Aspergillus terreus</i><i>Aspergillus nidulans</i>detection |
| spellingShingle | Maria Tokamani Eleftheria Figgou Lito Papamichail Eleni Sakka Athanasios Toros Anastasia Bouchorikou Antonis Giannakakis Efthymia Iliana Matthaiou Raphael Sandaltzopoulos A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five <i>Aspergillus</i> Species Journal of Fungi <i>Aspergillus fumigatus</i> <i>Aspergillus flavus</i> <i>Aspergillus niger</i> <i>Aspergillus terreus</i> <i>Aspergillus nidulans</i> detection |
| title | A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five <i>Aspergillus</i> Species |
| title_full | A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five <i>Aspergillus</i> Species |
| title_fullStr | A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five <i>Aspergillus</i> Species |
| title_full_unstemmed | A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five <i>Aspergillus</i> Species |
| title_short | A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five <i>Aspergillus</i> Species |
| title_sort | multiplex pcr melting curve analysis based detection method for the discrimination of five i aspergillus i species |
| topic | <i>Aspergillus fumigatus</i> <i>Aspergillus flavus</i> <i>Aspergillus niger</i> <i>Aspergillus terreus</i> <i>Aspergillus nidulans</i> detection |
| url | https://www.mdpi.com/2309-608X/9/8/842 |
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