PCR for Cryptosporidium detection?

Mark Siddall mes at zoo.toronto.edu
Sun Nov 27 21:20:02 EST 1994


In article <slaubac1-261194193212 at mac02.whartonab2.swarthmore.edu> slaubac1 at cc.swarthmore.edu (Stephen Laubach) writes:
>Dear bionetters,
>	
>	I'm doing a research proposal on a biological problem in a non-Western
>country for a microbiology seminar.   I chose to address detection of
>cryptosporidium in Costa Rica.  From one article I've read , Giardia cysts
>have been detected with a cDNA probe.  I imagine the same could be done
>with Cryptosporidium oocysts, but I haven't found any published articles
>(only immunoflourescence has been used).  Would a subunit of the 16s-like
>rRNA be used, as in Giardia?  What gene specifically?  As a backup plan, I

In short the 16S like gene should be fine.  We have had great success using 
the 16S gene for primer pairs for detecting protistan parasites in 
bivalves.  No reason why it shouldn't work for Crypto.
I would simply do a multiple alignment across Crypto and a variety of 
other taxa that might occur in the intestine from the phylum Apicomplexa.
For example a few Isospora species and Toxoplasma.  Then. based on the 
results, identify variable regions where the primers will only 
stick to Crypto keeping in mind that you want a 3' GG or CG or CC to help
with the sticking, you want about 50% GC, and you don't want more than
3 bases in a row that are the same.  After designing the primers,
send them as a search query to blast at ncbi.nlm.nih.gov to double check
against other known sequences.

Mark

-- 
Mark E. Siddall                "I don't mind a parasite...
mes at vims.edu                    I object to a cut-rate one" 
Virginia Inst. Marine Sci.                     - Rick
Gloucester Point, VA, 23062



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