To the microbiologists out there:
I'd like to measure total bacterial/fungal load in clinical samples by qPCR with published primers+ probes targeting the bacterial 16S/fungal 18SRNA gene. For standardization I'm going to create a plasmid standard curves with the PCR product of DNA from pure cultures
For the determination of the molecular weight of one copy, I need the sequence or at least exact length of the area between the primers. However, I cannot find the genome of the strains I'm working with, might be due to lack of knowledge about nomenclature or because it's not published so far?.
--> Do different strains of e.g. E coli or other microorganisms differ in sequence in the 16S/18S rDNA region? Or could I just use the sequence of another strain accessible via ncbi? Or do I necessarily have to sequence if I cannot find the sequence in the internet?
I work with:
- Candida albicans DSM 1386 (https://www.dsmz.de/collection/catalogue/details/culture/dsm-1386)
- Aspergillus fumigatus DSM 819 (https://www.dsmz.de/collection/catalogue/details/culture/DSM-819)
- common E. coli, K12, DH5alpha, thyA-
Thanks a lot!
Sarah
HI Sarah,
if you are interested I have a system for this quantification in a field test including standards. In case you are interested pls let me know
Sven
If you blast the primer sequences, don't you get hits? If you do, it's simple to get the amplicon length. It might be ok if this is not identical for different species. You can easily live with 10% error (it's hard to get more precise quantifications anyway, and the sampling variance is also usually much larger).
Giorgia Pertile To determine the number of copies present in the purified PCR product. I plan to measure the concentration of DNA with a spectrophotometer and knowing the molecular weight of one copy, one can calculate the number of copies from that.
Jochen Wilhelm Thanks a lot for sharing your experience! I do get hits if I blast and the sequence is similar from what I randomly compared, but sometimes the length varies, just a few bp though and definitely less than 10%. As I'm quite new to the field, that helped a lot!
Sven Reister Thanks for your offer; so far, I'll stick with the 'old' way, but I'll see.
For the calculatio you need the leght of your plasmid and your PCR- product and the concentration measure by nanodrop.
I send you the file I use to made this calculation.
Hi Sarah M. Volkers ,
I agree with Jochen Wilhelm . If you found the sequence, I strogly recommend gblocks gene fragments for qPCR standard curve. I am using them with good results.
site: https://www.idtdna.com/pages/education/decoded/article/tips-for-working-with-gblocks-gene-fragments
and
https://www.youtube.com/watch?v=Plkk8RfI4rA
Best,
Jacó
Jacó Joaquim Mattos : Just saw your answer, sounds very useful indeed!! I'll have look. Thanks!
Hi Sarah,
Please take also into account that the number of copies of 16S rRNA on the genome can vary across bacteria from different species (1 to 15). If you will experimentally contaminate your samples with your E. coli strain of interest, this difference is of course not relevant, but it can lead to misleading results if you have more complex microbial communities.
@Alessia Levante Thanks for your answer! I know, I read about it. Guess that's a problem that can't be solved... I'm going to normalize to copy number only and present the data that way as well (not talking about number of bacteria or anything like that) if it's going to work out at all. Guess I'll have to see how much of a difference between samples I'm going to find to decide if I can conclude anything from it... I agree, that can be a limiting factor!
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