I want to confirm the identity of some clinical isolates of E. coli, Staph. aureus, and S. typhi using PCR.
I am looking for a single pair of primers which can be used to produce PCR product of different lengths for the three different species. Preferably one where the amplicon length is already available in literature. I didn't find any such single pair in my search in Google Scholar and Pubmed.
Is there any such primer pair?
Hi there... quite ambitious assay you want to do.
Not done anything like this between different species, but one line of thought you might consider is 16S ribosomal RNA? 16S has regions that are specific across different species but also hypervariable regions that are species-specific...
Try looking up 16S sequencing or PCR on Pubmed - better than Google scholar...
Hope it helps!
Hello Shadid,
that should be really difficult. The only way would be to use 16s primers and sequence your pcr product. But that could be very difficult for E. coli and Salmonella because they are closely related. Why not use phenotypic methods or selective agar media?
Best regards
Dirk
Dear Dirk, I have already performed most of the phenotypic tests for Salmonella and E. coli. I was planning to confirm their identity through separate primer pairs (as available in Literature), but then I decided to test Staph. aureus too. I was really hoping to avoid buying three different primer pairs for identification only as it really stretches my budget [I plan to amplify another sequence in these bacteria related to chloramphenicol response]. Oh well..
Dear Poh, I am not clear about this hypervariable region... will I have to sequence such region to identify or will amplicon length be different enough for gel imaging?
Shadid,
You will need to download some 16S sequences and blast them together to find out the conserved regions and hypervariable regions. I've not done this before but I'm guessing you can design conserved primers that span the hvr so when you amplify you'd get different band sizes (as long as the hvr are different in size between species). otherwise it'll be a matter of sequencing the pcr products.
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