Dear Sepideh Mahboobi ! Greetings

From what you have written, I suppose you want to design specific primer that can amplify any bacteria under the wide population, available in the gut (for example, you want a primer that can detect and amplify any bacteria in firmicutes/bacteroidetes).

• If what I understood is right, you are in requirement of a universal primer for a wide population. If so, I would suggest you to target known universal sequences like 16S (present in almost all bacteria), since targeting them can amplify any species, and with sequencing the pattern of nucleotides, you can distinguish one species from another by matching for hits in BLAST.
• Another way, you can target any species finding the most commonly produced proteins in all bacteria and targeting the gene that encodes them, since the gene is essential for encoding the protein in all bacteria. But, also make sure that targeting a common region in bacteria should produce an amplicon which can be distinguished in nucleotide pattern for each bacterial species.
• If you want to target a very particular species, I suggest you to find any specific DNA for the species and design primers with specificity parameters.

All of the above mentioned can be performed in Primer BLAST or Primer3Plus.

Cheers!

16S rRNA amplicon sequencing followed by NGS.

Aravind Kumar N

Thank you Aravind for your time and comprehensive guidance. I will take your suggestions into account

Jetty Ramadevi Thank you so much but unfortunately I do not have access to analysis techniques and can not afford it although I have collected all stool samples from my patients.

If that is the case, I advice simple and easy method.

Whatever the samples you have, hope you can isolate DNA from them and you can amplify all samples with 16S rRNA then go for sequencing with whatever the facility available for you. If you have normal sequencing facility in you institute its fine. Otherwise generally people opt for Korea, by sending samples by FeDEx, cheaper. Check the following link, you get useful information about sequencing company etc.

(https://downloads.hindawi.com/journals/bmri/2020/3054195.pdf)

Once you receive the sequences assemble/pair Forward and Reverse, check the sequence if any abnormalities/excess of bases edit them. then save the assembled then blast in NCBI Nucleotide, you get all similar species match to that.

Hope it is clear.

Feel free to ask doubts.

with Best Wishes.

Aravind Kumar N

How to find species-specific DNA and design primers with specific parameters? I want to design specific primers for specific species. This is a website for finding unique fragments of different species (Insignia-DNA signature search server), but it seems to be closed. Do you have any other ideas to find specific genes or fragments?

Greetings Hang Liu

You can try Primer BLAST and Primer3Plus for designing primers with specific parameters. If you want to know about the parameters, a short handbook for understanding BLAST and their parameters is also made available by NCBI.

As for species-specific DNA, please find the complete cds of the species (For example, Ovis aries genome, complete cds). There you can find various gene present in the species. Try comparing the genes of one organism with atleast 10 other organisms which are closely related by BLAST to identify an unique gene.

Alternatively, you can do pick a gene in the organism of your interest (just make sure it is an essential nuclear genome and also it has lots of introns) and provide them in Primer BLAST. Try adjusting the nucleotide mismatches parmeters and Tm parameters and attempt so that in a particular set of parameters, your primer will amplify only the DNA which you have provided for input.

PS: These are all alternate and very effective methods when websites like Insignia is not present.

If you want further discussion, feel free to contact f so I can provide more elaborate explanation and additional methods.

Cheers!

Thank you Jetty Ramadevi so much.

I will study and search more and will ask for your help

thanks for your time