The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences provide varying degrees of success. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set.
The link below for an open access article with ITS-specific fungal primers:
Article Fungal-specific PCR primers developed for analysis of the IT...
You can use universal primers specific for amplification of ITS1-5.8S-ITS2 and 26S rDNA region using genomic DNA as a template. Primers are : ITS1 forward
(5'-TCCGTAGGTGAACCTGCGG-3') and ITS2 reverse (5'TCCTCCGCTTATTGATATGC-3'). They usually amplify ~450-550 bp fragment.
If still you are not sure about the species, you may go for ITS-RFLP.
You can try to combine the following commonly used primers in a nested PCR
1a) amplify a large fragment using ITS1F as the forward primer and LR5 or NL4 as reverse;
1b) amplify a large fragment using V9G and s the forward primer and LR5 or NL4 as reverse;
2) using a large fragment as the DNA-target, amplify small fragments using ITS1 and ITS4 for the ITS region, and NL1 and NL4 for D1/D2 domains of the LSU rRNA.
You can find sequences of the primers here:
https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/
https://en.wikipedia.org/wiki/Fungal_DNA_barcoding
Article Dual DNA Barcoding for the Molecular Identification of the A...
It depends on the region that you want to target and the molecular approach that you want to use. For example, if you are going to sequence your PCR amplicons with Illumina technology you can use primer pairs targeting the ITS1 or the ITS2 region, without the need of a nested approach.
For the ITS1 region the universal primers are ITS1F/ITS2R.
For the ITS2 there are many primer sets.
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