Fungal Identification by PCR-Sequencing (Sanger): Introduction, Principle, Clinical Significance, and Keynotes
Pure culture of Candida
Introduction
Table of Contents
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
Fig. Pure culture of Candida
DNA Extraction: Fungal DNA is extracted directly from pure culture or, in some cases, from clinical samples.
PCR Amplification: Specific primers target conserved regions flanking variable sequences, commonly:
D1/D2 domain of 28S rDNA – for yeasts and filamentous fungi.
Other loci (β-tubulin, calmodulin, actin, EF-1α) for improved resolution.
Sanger Sequencing: PCR products are purified and subjected to dideoxy chain-termination sequencing.
Sequence Analysis: Generated sequences are compared with reference databases (GenBank, CBS-KNAW, ISHAM Barcoding Database, UNITE) for species identification.
Fig. Clinical sample
Clinical Significance
Accurate Identification: Provides reliable species-level identification, especially for molds with overlapping morphology (Aspergillus, Fusarium, Scedosporium, Penicillium).
Drug Resistance Monitoring: Detects specific mutations in resistance-associated genes (e.g., ERG11, FKS1, CYP51A).
Outbreak Investigation: Useful in tracing infection sources and differentiating strains during hospital outbreaks.
Rare/Novel Fungi: Identifies fungi that are difficult or impossible to culture, including emerging pathogens like Candida auris.