I have isolated different species of Pseudomonas in a specific medium.
How can I identify them at species level by using universal primers or ITS primers?
Do ITS primers help in identification at species level?
I would think that if you are specially interested in Pseudomonas isolates, you can recheck your isolates with a Pseudomonas specific primer to make sure that they are all Pseudomonas before performing further analysis.
After that you can amplify 16s rRNA sequences with 27F and 1492R, if you can get a good sequences, it should be able to give you the species information by BLAST on NCBI or by matching sequences on Ribosomal Database Project.
The ITS sequences aren't very helpful when I try to match sequences on RDP, but does show the information about species with BLAST. But sequencing ITS sometime tricky because you might end up with multiple bands (due to difference in number of bp in each regions) which require cloning then sequences (which is an expensive process). I am actually waiting for the sequences of ITS after cloning coming back... I will let you know how it goes.
Anyway, I think it's a good idea to check if they are Pseudomonas first then amplify 16s rRNA. If you really want to see how do they related or difference in sequence, try with ERIC/BOX and MLST sequences.
A full length 16S rRNA gene sequence (27F +1492R primer pair) will be helpful for identification of bacterial species and Pseudomonas is a bacterial genus. ITS is more applicable for fungi. Checking if they are Pseudomonas or not is easy. Gram staining will give you some basis clues!
From gram staining, what are others information that you can have? beside knowing whether the bacteria are gram positive or negative and their shape. I did gram stain before but I am not sure if I retrieved all the information that it can provide
Thanh Nguyen - I mentioned about gram staining - because it is a very basic and useful technique to detect the gram characters and shape. So, a simple gram staining could be helpful - just to make sure if you have gotten isolates with Pseudomonas like gram character and shape.
Pavan Kumar Dumble - You first need to try sequencing the full 16s rRNA gene or 16S rDNA (primers - 27F +1492R) for each of your isolates. If that cannot resolve the problem of classifying them at the species level then you may also sequence the rpoB gene for each of these isolates. To further characterize at the strain level you may perform 'ribotyping'. There are other techniques to characterise bacterial isolates at species and strain levels.
Dear Chirajyoti ,
Thank you for reply , sir isolates which i have are pseudomonas for sure , the molecular detection is becoming difficult for me i wanted a primer which could identify at genus level , later the sequencing will give me identification at species level sir please share if you have papers regarding to pseudomonas molecular based identification
Thank you for your time
Dear Jai Ghosh,
Thank you reply , yes I get I need to identify with 16s RNA Sequence but I am getting trouble in sequence details if you have any paper regarding please attach me for reference
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Hi Pavan
Many references on attachment 1
I hope useful for you
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350142/
2
https://journal.frontiersin.org/article/10.3389/fmicb.2017.00413/full
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5
http://aem.asm.org/content/81/7/2579.full?maxtoshow=&hits=10&RESULTFORMAT=&fulltext=phylogenetic&searchid=1&FIRSTINDEX=200&resourcetype=HWFIG
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https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=8&cad=rja&uact=8&ved=0ahUKEwjqmKXV_tHUAhVPahoKHWKGDfoQFghNMAc&url=http%3A%2F%2Fwww.imedpub.com%2Farticles%2Fmolecular-characterization-of-pseudomonas-sp-isolated-from-milk-samples-by-using-rapdpcr.pdf&usg=AFQjCNFn9OuZ47WPJZRU0d6PNNI7Gtyn9A
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http://www.microbiologyresearch.org/docserver/fulltext/micro/133/3/mic-133-3-683.pdf?expires=1498155956&id=id&accname=guest&checksum=2F87AEA3221B317F793D2FC9584B957A
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https://onlinelibrary.wiley.com/doi/10.1046/j.1472-765X.1997.00377.x/pdf
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