I have extracted DNA from different species of staphylococci. I want to make a PCR and then to sequence the obtained product for staphylococcus species determination using BLAST search. However I would like to know what concrete primers should be used for the PCR i.e. for obtaining necessary product for sequencing. Can anybody recommend primers?
Http://www.ncbi.nlm.nih.gov/pubmed/24222453
http://www.ncbi.nlm.nih.gov/pubmed/24085691
http://www.ncbi.nlm.nih.gov/pubmed/23635328
Hello,
In my opinion primers targeting the tuf gene which encodes the elongation factor Tu are the best solution. I use them routinely during my experiments. Taking into account subtle differences among certain species I’d recommend you additional verification of ambiguous results with 16S rDNA sequencing and API Staph 32 phenotypic examination (however the latter one enables us to identify only 32 of 49 currently known staphylococcal species).
Here you have references for the primers I mentioned above:
Martineau F, Picard F, Ke D, Paradis S, Roy P, Ouellette P, Bergeron M. Development of a PCR assay for identification of staphylococci at genus and species levels. J Clin Microbiol. 2001. 39:2541-2547 - for tuf primers
htpp://rdna4.ridom.de- for 16S rDNA sequencing
Regards!
Thank you so much. There were 2 pairs of primers presented. Which pair should be used for determination of species?
I'd highly recommend you to use primers designated TStaG422 and TStaG465 that generate product of 370bp. In case of ambiguous results use primers 16S-27f and 16S-907r, that will generate amplicon about 880 bp, nevertheless amplification and sequencing of tuf is usually sufficient.
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