The genes for which you have designed your primers must be specific for that bacterial species. That is why you are getting the "text book" result. That I think is an evidence that your primers are specific for the bacterial species. Further, you can show that the primer you have designed does not recognise any part of a different bacteria. I do not know what would be other ways.

If you know the sequence of the region being amplified, a nucleotide BLAST search can give you the bacteria where an amplification can be observed. Also you can in silico analyze a target bacteria by BLAST.

You can do BLASTsearch with your sequence, or also you can use positive control  (with specific know bacteria specie)  in you PCR.

 please google the same sequence., if there is any literature regarding that sequence, you can find it very easily.,

and also you can find the genomic sequence in the PuMed also.

if you still doubt that., run the pcr with genome of different bacteria that you suspect for the cause of sepsis.

this only my hypothetical answer., 

For relatively low cost, you could have a library built from your PCR amplicons for sequencing by MiSeq or other NGS platform. This will let you see the diversity of amplicons that were generated by your primers. If there are multiple amplicons that are the same size on a gel, this will resolve the different sequences in that band.

This is actually quite a challenging problem and there is no one-size-fits-all solution. 

With the huge amount of sequence data available in GENBANK, a BLAST search is mandatory. However, do bear in mind that primers may have a certain degree of mismatch to the target and still cross-react. This is particularly true when the primer can form a G:T basepair, which is almost as stable as A:T. BLAST makes no distinction between the different kinds of mismatch, although some (eg purine:purine, pyrimidine:pyrimidine and gaps are highly destabilising. However, even such mismatches may be tolerated if they are at the 5' end of the primer.

Note also that BLAST will truncate the alignment if it hits a mismatch near the end of the sequence. For this reason, I usually BLAST with the whole amplicon, which prevents this happening. Changing the bases adjacent to the 3' ends of the primers is useful in order to mark the end of the primer.

You can exclude your target species from the BLAST search. This is useful.

Another, more traditional, approach is to test the primers against samples of other bacteria likely to occur in your sample material. This is relatively easy if you are looking at normally sterile material like urine, but it is virtually impossible if your sample material is faeces or soil. A variant on this is to test against samples of all close relatives of your target organism.

A glance through the materials and methods sections of articles on diagnostic PCR from the Journal of Clinical Microbiology is a good way to find out what is likely to be accepted by reviewers.  

Hello. You can also try an NCBI primer BLAST (see the link below) which rather than generally BLASTing against entire genomic regions/genes/everything, NCBI will show you the 'likely' templates that will be amplified. It's a dedicated version of BLAST for this purpose. It will show you where the primers are binding, and how well. This is a good indication of whether it is likely (or even possible) for your primers to bind any number of sequnces. I am currently doing something similar to determine how many species of Plasmodium the 'pan-Plasmodium' primers I am using really are.

P.S - just check the default settings before doing the BLAST, I think it defualts to Homo sapiens, so just remove that and change the 'database' drop-down list to 'nr' (meaning it will search against all catalogued sequnces, rather than a subset).

Hope this helps.

https://www.ncbi.nlm.nih.gov/tools/primer-blast/

prime Blast is your answer.

Hi Bradley, 

I looked at Primer Blast. As far as I can see, it only accepts a single accession as a target sequence to be investigated (though it might accept a list). 

I can see how this could be useful for checking specificity within a genome in order to stop you getting unwanted extra products, or even for a selection of potentially cross-reacting genomes, but I'm not entirely sure I see how it could be used to solve Prashant's problem. 

Could you clarify? 

Hi Andrew,

Whilst this is not by sequencing as Prashant first suggested, it should do the trick. Primer BLAST is dedicated to primer design and checking.

With Primer BLAST, you are correct that at the top it accepts a template/target sequence, but you can leave this box blank, fill in your forward and reverse primer sequence in the following boxes, and under the 'primer pair specificity checking parameters' section change the 'database' default selection from 'RefSeq mRNA' to 'nr'. 

Rather than comparing your pimers to a particular sequence of interest, when you click BLAST, this will now compare these specific primer sequences to the NCBI database and return all hits that are likely to be amplified. So, if they are truly specific, we should only see that particular bacteria being returned. If they are slightly less specific, we'll get more returned (and if non-specific we'll see all sorts of distantly related species returned). This provides confirmation in silico of how specific (or not) the primers are likely to be.

Of course, this is limited by what is in the database, and provides a likely indication, rather than experimental evidence (which would require testing the primers against a number of different, closely related bacterial species to see if they too are amplified). Generally, I would think that this is more than enough to get confidence in your primers (1) they are working on your specific bacteria, and 2) Primer BLAST returns only your bacteria of interest because the primers are species specific).

Hope this helps.

That's neat! Thanks, Bradley. I just tried it out and it seems to do just what I want it to. I've been looking for something like that for ages! 

Andrew