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Analyzing Vibrio parahaemolyticus diversity - (May/12/2010 )

Hey guys,
I'm working on a new project where I'm comparing species/strain diversity of the bacteria Vibrio parahemolyticus in different areas in a salt marsh and I'm looking for a method that will allow me to do this. I want to stay away from culture methods because if i want to look at the whole diversity i need to include to non-cultureable strains also. Any ideas? Thanks!

-scgradstudent-

There's been some success with species and strain identification by PCR amplifying and sequencing the 16s rRNA gene (conserved) and the 16s-23s intergenic spacer region adjacent to it (variable). The sequence of the 16s segment will give you the species ID and the sequence of the 16s-23s ISR can, in some species, differentiate species at the strain level.

-HomeBrew-

I've acutally came across a paper that analyzed the 16s-23s region for vibrio species. They mentioned in the paper that the primers they developed were vibrio specific, but when i primer-blast the primers, they hit pretty much everything. I was hoping to develop some type of DGGE protocol.

-scgradstudent-

Yes, I think you can make the species specific primers for your purpose if you want to apply 16S-23S PCR. I also recommend you DGGE but please note that Vibrio have several ribosomal operons that may not be identical and that cause false positive diversity on DGGE graphs. How do you think to use another marker like gyrB?

Good luck

-phucvn-

scgradstudent on May 13 2010, 12:29 PM said:

I've acutally came across a paper that analyzed the 16s-23s region for vibrio species. They mentioned in the paper that the primers they developed were vibrio specific, but when i primer-blast the primers, they hit pretty much everything. I was hoping to develop some type of DGGE protocol.


I guess it depends on what you mean by "hit pretty much everything". Sure, there will be BLAST identities with stretches of sequence from many species, but would these primers actually amplify from these species in a PCR? A single 3' base mismatch of either primer would cause the PCR to fail...

-HomeBrew-