I have isolated some fungi from soil and now I am trying to identify the species. For the identification what are the best primers (Universal/Other) to amplify fungal DNA and where I can get them? How many base pairs must I sequence for identification?
Please suggest me both forward and reverse primers which can able to amplify easily and to get the best results and also suggest some of the best reviews or articles related to fungi identification which are published in 2013/14
Thank you all
I have worked with may fungal pathogens and used White et al. 1990 primers for ITS1 and ITS4. These are working well. the sequence of these primers can be found on "White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and
direct sequencing of fungal ribosomal RNA genes for phylogenetics.
In: PCR protocols: a guide to methods and applications.
Academic Press, San Diego"
ITS Primers (White et al., 1990)
ITS1-F CTT ggT CAT TTA gAg gAA gTA A
ITS1 TCC gTA ggT gAA CCT gCg g
ITS2 gCT gCg TTC TTC ATC gAT gC
ITS4 TCC TCC gCT TAT TgA TAT gC
ITS5 ggA AgT AAA AgT CgT AAC AAg g
The "ITS" primer will amplify ITS region and not the 18S rRNA gene specifically but the 5.8S, 18S, 28S... So if the aim is to amplify 18S specifically, the ITS1 / ITS4 primer will not be good (even if they are excellent primer), unless you don't mind the region you amplify.
Please do a search on google/scholar, there is plenty of articles using fungal primer for 18S specifically!
See this webpage:
https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/
you could try, at first, using primers from White (1990) ITS 1, 2, 3 and 4.
A search in pubmed will lead you to hundreds of papers in which, each author uses their "personal" primers which are (of course) the best for their model... anyway, try with White´s primers...
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