I'm trying to design a system to trace a specific strain belonging to Enterobacter spp. in a bioaugmentation trial in anaerobic digesters
I have experience designing primer sets to bacterial targets.
By the wording of your question I assume you are looking for a particular strain of a given Enterobacter species. Or are you looking to be able to detect whether a given isolate you recover is of a particular species of Enterobacter? Or are you just looking for anything that looks like it might be an enterobacter?
These are very different questions.
If you are looking to check whether an isolate may be an enterobacter, then the genera specific primers in NCBI or the like will do the job. But beware, species move from genera to general in the taxonomic literature very frequently. Your answer might not be meaningful.
If you are looking to be able to detect a given strain of a given Enterobacter species amongst other similar strains things get more complicated and probably meaningful. Standard primers and rRNA genes will probably fail to give meaningful results and there may be no useful differences.
To do that, follow this methodology OR do MMLV OR just sequence the whole genome of your target isolate (it is cheap now)
1) Mine NCBI for any genomes of your target species and line them up with the software of your choice. This will give you some idea if what you are doing is meaningful or even possible. Assess the genetic diversity.
2) Design 10 primer sets that are dispersed on the genome and utilize the genetic diversity from the genome sequences. Design these primers based on every available sequence from that species in NCBI as there may be genetic diversity between isolates (or sequencing errors)
3) Do simple PCR, sybrGreen , or TAQMAN PCR on at least 50 independent isolates with the 10 primer sets.
4) Do linkage studies of your PCR targets to make a family tree. Now you have some knowledge about the actual differences between strains and have some chance of actually detecting and discriminating your target strain.
TAQMAN is very expensive, but it is specific and sensitive enough to give meaningful results on at least some crude unprocessed samples. Any processing may bias your results significantly
Use Allele ID software. http://www.premierbiosoft.com/bacterial-identification/
For RT-PCR primers I use the PrimerQuest tool on supplied by IDT-dna technologies
http://www.idtdna.com/PrimerQuest/Home/Index
For all other primers I use the free software called ApE DNA to generate primer sequences.
You can check their specificity using the primer-Blast tool on the Pubmed website selecting your Enterobacter genome if available
http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome
If you can differentiate your Enterobacter on 16S then ARB http://www.arb-home.de/ has all the tools you need to design FISH probes. For qPCR you will have use software designed to design primers .
Depending upon your platform I have booked succes on both fronts (FISH probes & qPCR primers) with Primrose, Oligocheck & Oligoreport (java jar files, cross platform). On a linux machine the above suggested ARB is still one of the best software packages around. You can acutally also check primer specifity with ARB. Depending on which database you use, it might get more (computationally) challenging for the tools outlined above. Therefore primer-blast http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome is probably the best suggestion. Another very good tool to design primers is Primer3 plus http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/ it allows for very extensive design criteria, however you will have to use one of the strategies above to evaluate specificity afterwards.
I think that you have enough tools to chose; anyway I recommend you to use primer 3, similar to Frederiek-Maarten suggest you. In most of my work I use it and I have gotten successful results.
I have experience designing primer sets to bacterial targets.
By the wording of your question I assume you are looking for a particular strain of a given Enterobacter species. Or are you looking to be able to detect whether a given isolate you recover is of a particular species of Enterobacter? Or are you just looking for anything that looks like it might be an enterobacter?
These are very different questions.
If you are looking to check whether an isolate may be an enterobacter, then the genera specific primers in NCBI or the like will do the job. But beware, species move from genera to general in the taxonomic literature very frequently. Your answer might not be meaningful.
If you are looking to be able to detect a given strain of a given Enterobacter species amongst other similar strains things get more complicated and probably meaningful. Standard primers and rRNA genes will probably fail to give meaningful results and there may be no useful differences.
To do that, follow this methodology OR do MMLV OR just sequence the whole genome of your target isolate (it is cheap now)
1) Mine NCBI for any genomes of your target species and line them up with the software of your choice. This will give you some idea if what you are doing is meaningful or even possible. Assess the genetic diversity.
2) Design 10 primer sets that are dispersed on the genome and utilize the genetic diversity from the genome sequences. Design these primers based on every available sequence from that species in NCBI as there may be genetic diversity between isolates (or sequencing errors)
3) Do simple PCR, sybrGreen , or TAQMAN PCR on at least 50 independent isolates with the 10 primer sets.
4) Do linkage studies of your PCR targets to make a family tree. Now you have some knowledge about the actual differences between strains and have some chance of actually detecting and discriminating your target strain.
TAQMAN is very expensive, but it is specific and sensitive enough to give meaningful results on at least some crude unprocessed samples. Any processing may bias your results significantly
thank you everybody for the suggestions and recommendations.
As Glenn asked, actually I'm trying to design primers and probes for 2 specific strains of E.cloacae and E.aerogenes. I have to track their survival in a complex sample in which it's very likely to find autochtonous closely related Enterobacter spp. strains.
This is the challenge!
You may give this tool a try.. http://www.microbial-ecology.net/probebase/
Luciano,
go to gene bank and search for your specific strain (both mRNA and genome), compare them to identify the splice junctions. mRNA message software (free online) will help you to remove spaves and numbers. design your forward and reverse primers to cross or involve at least one splice junction using primer 3 software (free online). make sure that melting temprature 2-3 degree C less than anealing temp and not far from house keeping gene. you can do the same with the probes but let it be about 10 degree C over the anealing temp. I prefare to use OneStep Quagene kit. Hopfully this information will help.
hello Luciano, do you solve your problem by now? Recently I have got the same problem as yours. I'm trying to make specific primer to identify a specie of Enterobacter. The 16s rRNA sequence data can not work for making specific primers, because these primers can also work for other bacteria species. Do you have any better suggestion for me to make specific primer for Enterobacter? Thank you and look forward to your reply. Jie
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