Fungal Molecular Diagnostics

PNA-FISH Basics

February 2011

This slide highlights the PNA FISH Yeast Traffic Light Probe method. The probes are single stranded peptide nucleic acids labeled with a fluorophore such as fluorescein or rhodamine. When a blood culture bottle signals as positive in the laboratory on an automated blood culture instrument, the technologist performs a Gram stain. If the Gram stain indicates that yeast are present, a small amount of the blood is then fixed to a fresh slide and the PNA FISH probes targeting unique rRNA sequences are added to the blood smear. The smear and probe combination is allowed to hybridize at 55°C for a specified amount of time and the slide is then washed to removed unincorporated probes. Finally the smears are mounted with a mounting medium and are examined by fluorescence microscopy.

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Jump to section:

• Introduction
• Objectives
• Traditional Methods of Fungal Diagnostics
• Traditional Methods
• Potential Advantages of Fungal Molecular Diagnostics
• Currently Available Molecular Diagnostics for Fungi
• Nucleic Acid Hybridization Probes³
• Nucleic Acid Hybridization Probes
• Nucleic Acid Hybridization Probes for Fungi
• Nucleic Acid Hybridization Probes
• DNA Sequencing for Fungi
• Ribosomal Structure: Fungi
• Traditional Sequencing Workflow
• Sequence Analysis
• Current Applications of D2 Fungal Sequencing at the Mayo Clinic
• Rapid, Direct Identification of Yeast from Blood Cultures
• PNA-FISH Basics
• PNA FISH for Candida spp—Direct Detection from Blood
• Yeast Identification Timeline from Positive Blood Bottle
• Potential Advantages of Direct Detection
• Fungal PCR Assays at Mayo Clinic
• Real-time PCR Workflow
• Pneumocystis PCR
• Pneumocystis LC-PCR Melt Curves
• Concordance of the Pneumocystis LightCycler PCR Assay and Calcofluor White Stain for BAL Fluid
• Coccidioides species Real-time PCR
• Melt-curve Analysis of Coccidioides spp Grown in Culture
• Utility of Coccidioides PCR Testing in the Clinical Setting⁴
• Meningeal Coccidioidomycosis Diagnosed by Real-Time PCR Analysis of Cerebrospinal Fluid⁵
• Histoplasma capsulatum/Blastomyces dermatitidis Real-Time PCR
• What lies in the future for fungal molecular diagnostics?
• Mass Spectrometric Identification Of Fungi
• Summary
• References
• Questions?
• Disclosure