Question: Bacterial identification - PCR primers - unknown target
0
gravatar for RAZLICAK
4.7 years ago by
RAZLICAK0
Serbia And Montenegro
RAZLICAK0 wrote:

Dear all, 

I obtained the job and need to identify presence of some bacteria in clinical samples by PCR with species/strain specific primers. The primers are already available in the lab, but the person who designed/find/order the primers were not available in the lab anymore and nobody knows the details. Therefore, I  know primer sequences, the names of the strains,  but I do not know the target (genome, 16S, ...), the size of the amplicon  - actually I have no data to check the PCR result... I tried to blast them, but only one pair is found on the NCBI. 

How to find the target sequences, can anyone help me. Are there are softwares or databases for blasting the bacterial genome/transcriptome? 

 

blast alignment sequence • 2.8k views
ADD COMMENTlink modified 4.7 years ago by Garan600 • written 4.7 years ago by RAZLICAK0
3
gravatar for Garan
4.7 years ago by
Garan600
United Kingdom
Garan600 wrote:

They might be MLST specific primers, maybe check the various MLST schemes on http://www.mlst.net/. If you could give the strain / organism names and an example primer sequence it would help.

ADD COMMENTlink written 4.7 years ago by Garan600
2
gravatar for mark.ziemann
4.7 years ago by
mark.ziemann1.2k
Australia/Mebourne/Geelong/Deakin
mark.ziemann1.2k wrote:

Which blast did you use? I hope you used microbial blast at NCBI.

If you know the strain and which primers are forward and reverse, then you could use primer blast to identify the target seq.

If that fails, go to UCSC genome browser for your species of interest and try in silico PCR.

If that fails, then download the reference sequence and use BLAT with the forward and reverse sequences concatenated.

If that fails, use agrep or similar to find the primer sequence in the unwrapped fasta file and bes sure to query the reverse complement as well.

If that fails, then you're looking in the wrong species and should probably order your own primers.

ADD COMMENTlink written 4.7 years ago by mark.ziemann1.2k
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