Does it possible to do colony PCR for filamentous fungi using the spores for identification through Universal Primers ITS 1 and 4.
thanks in advance
HI Bhavya: Yes, you can, if you got enough amount of DNA in good quality.
Yes, I agree with Oadi, the most difficult aspect will be obtaining a high-quality DNA extraction from spores, as it can sometimes be difficult even starting with fungal hyphae. The extraction protocol will probably need to include a mechanical disruption such as bead-beating in order to adequately break open the fungal cell walls.
It depends on the species of fungus. I had it work with a weft of mycelium and spores of Fusarium spp., but not with any other spp. As with pure DNA, melanin (polyphenols) and polysaccharides produce in culture will inhibit PCR. Temporary DNA preps from colonies can help in dilute inhibitors and increase DNA release. Pre-lysing the mycelia or spores using microwaves (http://www.fgsc.net/fgn43/ferreir.html) or bead-beating in excess volume (e.g. 10 ul fungus to 1 ml volume) of TE or water, can help increase DNA release and PCR success.
Hello Bhavya
You can find some useful references on attachment 1
http://cdn.intechopen.com/pdfs/34844/intech-molecular_tools_for_detection_of_plant_pathogenic_fungi_and_fungicide_resistance.pdf
3
http://bookshop.europa.eu/fr/fungal-identification-techniques-biotechnology-1992-94--pbCGNA16510/downloads/CG-NA-16-510-EN-C/CGNA16510ENC_001.pdf;pgid=y8dIS7GUWMdSR0EAlMEUUsWb0000Ykkn1avC?FileName=CGNA16510ENC_001.pdf&SKU=CGNA16510ENC_PDF&CatalogueNumber=CG-NA-16-510-EN-C
6
https://shodhganga.inflibnet.ac.in/jspui/bitstream/10603/136806/9/09_chapter_4.pdf
8
https://cfpub.epa.gov/si/si_public_file_download.cfm?p_download_id=461353
12
https://publicationslist.org/data/torsten-bohn/ref-55/Pallez_FdTEchBiotech_2014.pdf
13
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160356
14
http://onlinelibrary.wiley.com/doi/10.1111/j.1472-765X.2010.02907.x/pdf
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology