Development of multiplex real-time PCR for rapid identification and quantitative analysis of Aspergillus species

Autor: Chulmin Park, Sung-Yeon Cho, Won-Bok Kim, Hye-Sun Chun, Dong-Gun Lee
Jazyk: angličtina
Rok vydání: 2020
Předmět:
0301 basic medicine
Azoles
Antifungal Agents
Artificial Gene Amplification and Extension
Pathology and Laboratory Medicine
Polymerase Chain Reaction
law.invention
Aspergillus fumigatus
Database and Informatics Methods
Cytochrome P-450 Enzyme System
law
Medicine and Health Sciences
Multiplex
DNA extraction
Polymerase chain reaction
Fungal Pathogens
Multidisciplinary
biology
Physics
Fungal genetics
Eukaryota
Melting
Condensed Matter Physics
Real-time polymerase chain reaction
Aspergillus
Aspergillus Fumigatus
Fungal Molds
Medical Microbiology
Research Design
Physical Sciences
Medicine
Pathogens
Phase Transitions
Sequence Analysis
Research Article
Bioinformatics
Science
030106 microbiology
Mycology
Microbial Sensitivity Tests
Research and Analysis Methods
Real-Time Polymerase Chain Reaction
Microbiology
Fungal Proteins
03 medical and health sciences
Extraction techniques
Species Specificity
Drug Resistance
Fungal
Multiplex polymerase chain reaction
Aspergillosis
Molecular Biology Techniques
Microbial Pathogens
Molecular Biology
DNA Primers
Organisms
Fungi
Quantitative Analysis
Biology and Life Sciences
biology.organism_classification
Molecular biology
Ascomycetes
030104 developmental biology
Multiplex Polymerase Chain Reaction
Invasive Fungal Infections
Zdroj: PLoS ONE
PLoS ONE, Vol 15, Iss 3, p e0229561 (2020)
ISSN: 1932-6203
Popis: The identification of Aspergillus species and azole resistance is highly important for the treatment of invasive aspergillosis (IA), which requires improvements in current fungal diagnostic methods. We aimed to develop multiplex real-time PCR to identify major Aspergillus section and azole resistance. BenA and cyp51A genes were used to design primers, probes, and control DNA for multiplex PCR. Qualitative and quantitative analysis was conducted for 71 Aspergillus and 47 non-Aspergillus isolates. Further, the limit of detection (LOD) and limit of quantitation (LOQ) from hyphae or conidia were determined according to the culture time. Newly developed real-time PCR showed 100% specificity to each Aspergillus section (Fumigati, Nigri, Flavi, and Terrei), without cross-reaction between different sections. In quantitative analysis of sensitivity measurements, LOD and LOQ were 40 fg and 400 fg, respectively. Melting temperature analysis of the cyp51A promoter to identify azole resistance showed temperatures of 83.0 ± 0.3°C and 85.6 ± 0.6°C for susceptible A. fumigatus and resistant isolates with TR34 mutation, respectively. The minimum culture time and fungal colony size required for successful detection were 24 h and 0.4 cm in diameter, respectively. The developed multiplex real-time PCR can identify common Aspergillus sections quantitatively and detect presence of the TR34 mutation. Further, this method shows high sensitivity and specificity, allowing successful detection of early-stage fungal colonies within a day of incubation. These results can provide a template for rapid and accurate diagnosis of IA.
Databáze: OpenAIRE
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