Molecular detection and identification ofAspergillusspp. from clinical samples using real-time PCR
| Autor: | A.I. Aller, Jose C. Palomares, Mercedes Ramírez, Carmen Barroso Castro, Estrella Martín-Mazuelos, Javier Aznar, Ma José Torres, Maite Ruiz |
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| Rok vydání: | 2009 |
| Předmět: |
Aspergillus flavus
Dermatology Aspergillosis Polymerase Chain Reaction Sensitivity and Specificity Aspergillus fumigatus Microbiology law.invention Species Specificity Aspergillus nidulans law medicine Humans Aspergillus terreus DNA Fungal Mycological Typing Techniques skin and connective tissue diseases Polymerase chain reaction Aspergillus biology Aspergillus niger General Medicine biology.organism_classification medicine.disease Infectious Diseases Fungemia |
| Zdroj: | Mycoses. 52:129-134 |
| ISSN: | 1439-0507 0933-7407 |
| DOI: | 10.1111/j.1439-0507.2008.01548.x |
| Popis: | The definite and rapid diagnosis of invasive aspergillosis is necessary because of the high mortality caused. The objective of this study was to evaluate a real-time PCR assay to detect Aspergillus spp. in clinical samples, based on the Light Cycler technology. Specificity was assessed by using DNA extracted from pathogenic and non-pathogenic bacteria/fungi from Spanish Collection including: two Aspergillus flavus, four Aspergillus fumigatus, two Aspergillus nidulans, two Aspergillus niger and two Aspergillus terreus isolates. The analytical sensitivity was evaluated with different inocula (10(1)-10(5) conidia ml(-1)), and serially diluted DNA of A. fumigatus. To assess clinical applicability, samples from patients at risk were analysed. Species identification was determined by analysing the melting curves. Reactions using genomic DNA from other species of different genera than Aspergillus were negative (specificity: 100%). Analytical sensitivity was 60 fg using DNA and 5-20 conidia using conidial suspensions. The linear range was from 60 to 6 x 10(7) fg. The Tm ranged from 67.34 to 70.7 degrees C for the different Aspergillus spp. studied. Nine hundred and forty-eight consecutive blood samples from 127 patients were processed. In total, 10 (1%) of 948 samples from blood samples were PCR-positive. The real-time PCR assay provides a high sensitivity and specificity for detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species. |
| Databáze: | OpenAIRE |
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