Experimental evaluation of two ITS couples of primers for the determination of the fungal diversity.

Identification of fungal species for diagnostic or research purposes is a real challenge given their diversity. The current barcoding method recommends the use of ITS 1-2 primers (ITS1 region) for identification of clinical isolates. In metataxonomic study using new generation sequencing, ITS1-2 are the most frequently used to evaluate the diversity of fungi in floras and determined the so -called “mycobiome”. Given the diverging, results obtained in mycobiome studies using these primers, it is unclear whether these primers or which primers are adapted to uncover accurately and reproducibly the fungal diversity in such studies? Here, we want to analyse the parameters influencing ITS amplification in order to select the best couples of primers able to describe fungal diversity accurately using qPCR and SYBR Green experiments and standardized fungal DNA of 96 fungal species. 

We took advantage of the collection of clinical isolates handled at the French National Reference Center for Invasive Mycoses and Antifungals (NRCMA) and strains from international collection (CBS-KNAW Collection, Utrecht, the Netherlands), to select 96 fungal species representing the fungal diversity uncovered in clinical samples. Extraction procedure and DNA quantification methods were first evaluated. All DNA were standardized at 1ng/ml and screened using primers ITS 1-21 and 4-7 using the LightCycler® 480 instrument (Roche). Quantification cycles (Cq) and fusion temperature (Tm) were our endpoints for all experiments. In view of the strains commonly found in patient samples, mixtures of 2 DNA (from 6 species of filamentous fungi and 6 species of yeast) were tested and analysed according their Cq and Tm values.

ITS1-2 primers and ITS4-7 were not able to amplify all fungal DNA. The spectrum of amplification was different in both targets, with an advantage for ITS4-7 that amplified more DNA than ITS1-2. The Tm and the quantification was impacted using mixtures of DNA and ITS1-2 with almost all mixtures that gave aberrant results in terms of Tm. For ITS4-7, amplification of both DNA of mixtures was reliable but the amplicon with the highest Tm was preferentially amplified.

These preliminary results suggest that results provided by ITS1-2 and 4-7 amplification may not be adapted to investigate accurately the fungal diversity present in floras. And a diagnosis of a complex sample of several strains may be misjudged.