Hello scientists,
we isolated in our lab a nice promising biocontrol agent (Bacteria), now in field application, we need to re-isolated it and somehow make sur is the right one, we are thinking to go for a PCR detection approach, but to design a specific primers for a specific target ( strain-level) is not easy. I am wondering if you have any idea, how we can do such a thing ? knowing that bacteria was identified ( Pseudomonas synxantha) and we may have the whole sequence in near future.
Thanks in advance .
You could just immediately go to 16S sequencing. But once you have some strain specific genome data you might be able to find a unique gene or region to which you can design primers that would be specific. Obviously it is going to be harder to distinguish between your specific strain and some other closely related strains.
The other possibility is to introduced some marker or barcode into your strain, if that is permissible for the experiments you are doing.
Dear Fares,
You may be able to use PCR with 16S primers to determine relative numbers of your strain from field samples, but this might be difficult if Pseudomonas synxantha is common and if there is significant levels of intra-species diversity (i.e., high background levels means that you cannot distinguish your strains from other strains of the same species). We use a P. fluorescent strain and there is a significant level of genetic difference between strains in this species, so 16S species-specific PCR is not very helpful in trying to assess numbers in field experiments.
It might be more useful to directly determine CFU numbers by plating onto selective media such as PSA-CFC – especially if your strain has an unusual colony colour or morphology compared to the native pseudomonas (unlikely but possible).
Your next option would be to label your strain (noting Michael's warning above regarding regulations) and here you have two options – mark your strain randomly using a transposon-delivered antibiotic marker such as Mini-Tn5 Kanamycin-resistance (this is very effective when you use PSA plus Kan) or introduce the marker at a specific site (e.g., the Tn7 attachment site using a mini-Tn7 construct). These can be very effective, but you may need to demonstrate first that the carriage of the marker does not incur a fitness cost and you have to accept that some lineages of your strain may have lost the marker during the field experiment.
Zang and Rainey (2007) (10.1016/j.mimet.2007.07.001) developed a neutral marker for P. fluorescens SBW25 specifically to avoid fitness costs, but this required finding a region of the genome (a defective prophage site) that could be used to insert the marker without effecting gene expression.
Andrew
Fares Bellameche Such task was solved in many cases by arbitrary PCR some 20-30 years ago. Single adapter AFLP (sAFLP) is probably the best way to generate strain-specific markers and turn it into SCAR marker. sAFLP bands can be separated in agarose gel, and easily sequenced.
Hester J. Bootsma, Han G. J. van der Heide, Simone van de Pas, Leo M. Schouls, Frits R. Mooi, Analysis of Moraxella catarrhalis by DNA Typing: Evidence for a Distinct Subpopulation Associated with Virulence Traits, The Journal of Infectious Diseases, Volume 181, Issue 4, April 2000, Pages 1376–1387, https://doi.org/10.1086/315374
Dear Fares, I think 16S RNA is a good choice, fast and reliable.
On the other hand, you can also try biochemical tests depending on their nearest species
Michael J. Benedik, @Andrew J Spiers
Thank you so much for your kind suggestions, and definitely, it's not easy to get a primer for a specific strain. for marker, since it's a biocontrol agent that will be used in biopesticide formulation in an European project, I don't think, we can tag the strain with any kind of molecular marker.
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