Target Genes, Primer Sets, and Thermocycler Settings for Fungal DNA Amplification

This document describes some of the target genes and primers that can be used for DNA sequence-based identification of fungi and the PCR conditions with which to use those primers. Other primer sets have been used for other genes, but those described below are the most consistently available in databases for the identification of yeasts and molds that are most likely to be identified in a clinical microbiology laboratory.

Universal primer sets exist, but they often do not have enough discriminatory power to identify species, or they do not have the discriminatory power to identify species within a species complex, often giving 100% match to multiple species.

This tool assumes that fungal DNA already exists; it does not describe the procedure for purification of fungal DNA.

PCR primers and purposes

ITS

In general, for unknown molds, the ITS region of the rDNA is used as the primary target with primers ITS-1 and ITS-4 as the most general primer set. In some cases, these primers may not provide sufficient identification, and a protein coding region may be required.
For the forward primer there are two options. ITS-5 gives a slightly longer PCR product than ITS-1, but both are good.

Forward:               ITS-1 5’-TCCGTAGGTGAACCTGCGG

(or) ITS-5 5’-GGAAGTAAAAGTCGTAACAAGG

Reverse:               ITS-4 5’-TCCTCCGCTTATTGATATGC

Annealing temperature: 52°C

These primers amplify approximately 600 basepairs of the ITS1-5.8S-ITS2 region of the ribosomal cistron.

D1-D2 region of large ribosomal subunit

Although the ITS primers are universal for fungi, the D1D2 region of the large ribosomal subunit has better discrimination for yeasts, with primers NL-1 and NL-4.

Forward:               NL-1 5’-GCATATCAATAAGCGGAGGA

Reverse:                NL-4 5’-TTGGTCCGTGTTTCAAGACG

Annealing temperature: 52°C

These primers amplify approximately 620 basepairs of the 28S region of the ribosomal cistron.

Elongation Factor-1α (for sequencing Fusarium species)

The ITS primer set generally only discriminates Fusarium species into the various species complexes but does not discriminate cryptic species. For identification of Fusarium isolates within species complexes, the EF-1α primers should be used (O’Donnell, 2009).

PCR Forward: EF-1       5’-ATGGGTAAGGARGACAAGAC

PCR Reverse: EF-2       5’-GGARGTACCAGTSATCATG

Annealing temperature: 52°C

These primers amplify approximately 717 bp of the coding region of the EF-1α gene.

β-tubulin (for sequencing Scedosporium, Aspergillus and Penicillium species)

Similar to Fusarium, the ITS primer set generally only discriminates Scedosporium, Apsergillus, and Penicillium species into the various species complexes but does not discriminate cryptic species. For identification of Scedosporium, Aspergillus, and Penicillium isolates within species complexes, the β-tubulin primers should be used (Glass, 1995).

Forward:           Bt2a     5’-GGTAACCAAATCGGTGCTGCTTTC

Reverse:           Bt2b     5’-ACCCTCAGTGTAGTGACCCTTGGC

Annealing temperature: 54°C

These primers amplify approximately 495 bp of exons and introns at the 5’ end of the β-tubulin gene.

Dermatophyte primers (amplify the ribosomal ITS region of dermatophytes)

Trichophyton species DNA is amplified very poorly by the ITS primer set used for most other molds. There is a special set of ITS primers specifically for amplification of the ITS region of dermatophytes, especially Trichophyton (Gräser, 2000).

Forward:           LR1        5’-GGTTGGTTTCTTTTCCT

Reverse:           SR6R 5’-AAGTAAAAGTCGTAACAAGG

Annealing temp: 52°C

These primers amplify approximately 630 basepairs of the ITS1-5.8S-ITS2 region of the ribosomal cistron.

Trichosporon primers (amplify IGS)

Individual species of Trichosporon are not well discriminated using either the ITS or D1D2 primer sets. For identification of Trichosporon species, the intergenic region of the ribosomal cistron is used (Sugita, 2002).

Forward:           26SF 5′-ATCCTTTGCAGACGACTTGA

Reverse:           5SR      5′-AGCTTGACTTCGCAGATCGG

Annealing temp: 56°C

These primers amplify a section of the intergenic spacer in the ribosomal cistron. The sequence lengths vary greatly from approximately 200 basepairs to up to 700 basepairs depending on the species.

The Fusarium ID database:

The Fusarium ID database contains EF1-α sequences for many species of Fusarium.

References

Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 1995;61:1323-30.

O’Donnell K, DA Sutton, MG Rinaldi, et al. Novel multilocus sequence typing scheme reveals high genetic diversity of human pathogenic members of the Fusarium incarnatum–F. equiseti and F. chlamydosporum species complexes within the United States. J Clin Microbiol 2009;47:3851-3861.

Gräser Y, Kuijpers AF, Presber W, de Hoog GS. Molecular taxonomy of the Trichophyton rubrum complex. J Clin Microbiol. 2000, 38:3329-3336.

Sugita T, Nakajima M, Ikeda R, et al. Sequence analysis of the ribosomal DNA intergenic spacer 1 regions of Trichosporon species. J Clin Microbiol 2002;40:1826-1830.

Clinical and Laboratory Standards Institute. MM18-Ed2. Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing. Clinical and Laboratory Standards Institute, Wayne, PA. 2018.

Disclaimer:

CDC’s Mycotic Diseases Laboratory developed this document as a tool for choosing primers for DNA sequencing-based identification of fungi. It is the responsibility of the testing laboratory to ensure content and format are modified as necessary to meet applicable regulatory requirements, quality management system standards, and chemical and biological safety requirements. This is not a controlled document and the described test methods are subject to change without notice. It is the responsibility of the testing laboratory to ensure the information within this document remains applicable. Contact the Mycotic Diseases Laboratory (mdblab@cdc.gov) to find out whether any changes have been made.

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