Molecular Diagnostics: PCR and Sequencing

Molecular methods detect and identify fungi by their nucleic acids rather than by growth or appearance. Polymerase chain reaction amplifies fungal DNA from specimens or isolates, and sequencing of conserved regions, especially the internal transcribed spacer of the ribosomal gene cluster, reads the amplified DNA to name the organism, often faster and more precisely than culture.

Definition

Molecular diagnostics for fungi are techniques that amplify and analyse fungal nucleic acids, using PCR to detect fungal DNA and DNA sequencing of conserved regions such as the internal transcribed spacer to identify the organism to genus or species.

Scope

This topic covers species-specific and broad-range (panfungal) PCR, real-time PCR, DNA sequencing of barcoding regions such as the ITS, and the use of these methods on culture isolates and directly on clinical material, including formalin-fixed tissue. It frames them as identification and detection methods and offers no testing or treatment instructions.

Core questions

• Is fungal DNA present in this specimen, and from which organism does it come?
• When is broad-range panfungal PCR preferable to a species-specific assay?
• Which genomic region best discriminates the fungi of interest?
• How are molecular results interpreted given the risks of contamination and of detecting non-viable DNA?

Key concepts

• Polymerase chain reaction (PCR)
• Species-specific versus broad-range (panfungal) PCR
• Real-time (quantitative) PCR
• Internal transcribed spacer (ITS) as a fungal barcode
• DNA sequencing and reference databases
• Detection on isolates versus direct on tissue
• Contamination control and interpretation of cell-free DNA

Mechanisms

PCR uses primers and a thermostable polymerase to amplify target DNA through repeated cycles, generating enough copies to detect even small amounts of fungal genetic material. Species-specific assays target a known organism, while broad-range or panfungal assays use primers against conserved regions shared by many fungi, after which sequencing reveals the identity. The internal transcribed spacer region of the ribosomal RNA gene cluster, flanked by conserved primers described by White and colleagues, is variable enough between species to act as a universal fungal barcode, and the read sequence is matched against reference databases to name the organism. Real-time PCR adds quantification and speed, and assays can be run on cultured isolates or directly on clinical specimens, including formalin-fixed tissue, though degraded DNA and environmental contamination complicate interpretation. Because PCR detects DNA rather than viable cells, results are read together with culture, microscopy, and antigen findings.

Clinical relevance

Molecular detection and sequencing increasingly underpin how fungi are identified and how some infections are recognised, and certain PCR results contribute to consensus categories of probable invasive fungal disease. This entry describes the methods and their interpretive caveats as reference material and does not direct testing or patient care.

Evidence & guidelines

Consensus definitions from the EORTC/MSGERC incorporate specified PCR results among the mycological criteria for probable invasive fungal disease, reflecting the maturation of molecular methods, while best-practice recommendations describe their place alongside culture and antigen testing. The ITS sequencing approach traces to the widely used primers published by White and colleagues.

History

After the polymerase chain reaction was developed in the 1980s, its application to fungi advanced rapidly when White and colleagues published, in 1990, primers for amplifying and directly sequencing fungal ribosomal RNA genes, establishing the ITS region as a phylogenetic and later diagnostic marker. Real-time PCR, broad-range panfungal assays, and high-throughput sequencing subsequently extended molecular mycology from research into routine identification and detection.

Seminal works

• white-1990
• donnelly-2020

Frequently asked questions

What is the ITS region and why is it used to identify fungi?

The internal transcribed spacer (ITS) is a part of the fungal ribosomal gene cluster that varies enough between species while being flanked by conserved sequences, making it a practical universal barcode for sequencing-based fungal identification.

Does a positive fungal PCR prove active infection?

Not on its own. PCR detects fungal DNA, which can persist after organisms are no longer viable or arise from contamination, so molecular results are interpreted alongside culture, microscopy, and antigen tests and within standardised diagnostic definitions.