ArticlePloS one2020
Development of multiplex real-time PCR for rapid identification and quantitative analysis of Aspergillus species.
Article in PloS one, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed.
- A fully integrated sample-to-answer molecular diagnostic platform for rapid identification of four majorFrontiers in microbiology · 2026Article
- Distinction BetweenJournal of fungi (Basel, Switzerland) · 2025Article
- Recent developments inMicrobiology and molecular biology reviews : MMBR · 2025Review
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- Research Progress on Detection of Pathogens in Medical Wastewater by Electrochemical Biosensors.Molecules (Basel, Switzerland) · 2024Review
- Evaluation of Primers OPF-01, P54, and 1253 to IdentifyPathogens (Basel, Switzerland) · 2024Article
- Comparative proteomics analysis ofFrontiers in microbiology · 2024Article
- Article
- Clinical Implementation ofJournal of fungi (Basel, Switzerland) · 2023Article
- Article
- Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis.Genes · 2022Review
- Quantitative PCR Effectively Quantifies Triazole-Susceptible and Triazole-ResistantJournal of fungi (Basel, Switzerland) · 2022Article
- Practice Guidelines for the Diagnosis of COVID-19-Associated Pulmonary Aspergillosis in an Intensive Care Setting.Journal of intensive care medicine · 2022Review
- TheFrontiers in microbiology · 2021Article
- Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections.Journal of fungi (Basel, Switzerland) · 2020Article
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Authors and funding
5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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