Fungal Identification by PCR-Sequencing (Sanger): Introduction, Principle, Clinical Significance, and Keynotes

Introduction

Accurate identification of fungi is critical for clinical, environmental, and epidemiological purposes. Traditional culture and morphology-based methods are time-consuming and may misidentify cryptic or closely related species. PCR amplification followed by Sanger sequencing of conserved genetic loci (e.g., ITS, 18S rRNA, 28S rRNA, β-tubulin, actin, calmodulin) has become the gold standard molecular method for fungal identification. This technique provides precise species-level identification, especially for uncommon, novel, or drug-resistant fungi.

Principle

1. DNA Extraction: Fungal DNA is extracted directly from pure culture or, in some cases, from clinical samples.
2. PCR Amplification: Specific primers target conserved regions flanking variable sequences, commonly:
   • Internal Transcribed Spacer (ITS) – universal fungal barcode region.
   • D1/D2 domain of 28S rDNA – for yeasts and filamentous fungi.
   • Other loci (β-tubulin, calmodulin, actin, EF-1α) for improved resolution.
3. Sanger Sequencing: PCR products are purified and subjected to dideoxy chain-termination sequencing.
4. Sequence Analysis: Generated sequences are compared with reference databases (GenBank, CBS-KNAW, ISHAM Barcoding Database, UNITE) for species identification.

Clinical Significance

1. Accurate Identification: Provides reliable species-level identification, especially for molds with overlapping morphology (Aspergillus, Fusarium, Scedosporium, Penicillium).
2. Drug Resistance Monitoring: Detects specific mutations in resistance-associated genes (e.g., ERG11, FKS1, CYP51A).
3. Outbreak Investigation: Useful in tracing infection sources and differentiating strains during hospital outbreaks.
4. Rare/Novel Fungi: Identifies fungi that are difficult or impossible to culture, including emerging pathogens like Candida auris.
5. Limitations:
   • Requires pure DNA (contaminants can interfere).
   • More expensive and slower than MALDI-TOF.
   • Interpretation depends on the quality of the databases.

Keynotes

1. PCR–Sanger sequencing remains the reference molecular method for fungal identification in research and clinical mycology.
2. The ITS region is the primary universal fungal barcode, but additional loci improve species-level resolution.
3. Provides higher accuracy than morphology or biochemical tests.
4. Essential for identifying rare, cryptic, and resistant fungi in high-risk patients.
5. Best used in combination with culture, antifungal susceptibility testing, and clinical data for comprehensive diagnosis.
6. Though NGS is emerging, Sanger sequencing remains widely used due to reliability and accessibility.

Further Readings

1. https://www.cd-genomics.com/microbioseq/resource-rapid-bacterial-fungal-pathogen-identification-using-sanger-sequencing.html
2. https://www.slideshare.net/slideshow/fungal-identification-by-sanger-sequencingpptx/261928388
3. https://pmc.ncbi.nlm.nih.gov/articles/PMC2845394/
4. https://pmc.ncbi.nlm.nih.gov/articles/PMC88253/
5. https://www.scirp.org/journal/paperinformation?paperid=118671
6. https://www.cd-genomics.com/blog/sanger-sequencing-introduction-principle-and-protocol/
7. https://pmc.ncbi.nlm.nih.gov/articles/PMC10643650/
8. https://microbenotes.com/sanger-sequencing/
9. https://www.cd-genomics.com/blog/sanger-sequencing-introduction-principle-and-protocol/
10. https://testguide.labmed.uw.edu/view/FUNDNA
11. https://pmc.ncbi.nlm.nih.gov/articles/PMC12233950/
12. https://www.sciencedirect.com/topics/immunology-and-microbiology/sanger-sequencing
13. https://www.sciencedirect.com/topics/medicine-and-dentistry/sanger-sequencing