I have done a metagenomic assessment (Mi-Seq) for the interior bacteria of blowfly (Chrysomya megacephala) and got the results. At the moment, i need to verify the abundance of some species of bacterial genera such as; Wolbachia, Klebsiella, Dsygonomonas, ...etc through qPCR method (ABI PRISM 7000). Does anybody know what is the best way to get that primers? What are the variable regions that should be targeted to assess density of that bacterial species?
Many thanks in advance,
Hello Abdelaziz, let me give you the answer provided 6 years ago by Cassandra Kotter, for a similar question, which I have found very useful and helpful.
The 16s gene contains many variable and conserved regions. Ideally you will lay your primers down on a conserved region to capture as many bacterial species as possible. You then may want to amplify your product through a variable region to obtain information on species composition. There are several standard 16s primers available today and most researchers tend to use similar combinations of primers to target the 16s gene. Some of these include 27F, 338R or 515F, 806R. I have pasted some useful links below. The first is a great review on species-specific 16s primers. The second is actually a link to wikipedia - I know that seems awful - but wikipedia has a great summary of the different 16s primers including the sequences, which you can just paste into the IDT website to order them. The third link is a summary qPCR results with various 16s primers.
1) http://www.sciencedirect.com/science/article/pii/S0167701203002276
2) http://en.wikipedia.org/wiki/16S_ribosomal_RNA
3) http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2004.02409.x/full
If you find this helpful, I have a ton of other papers on individual sets of 16s primers, but I thought you might a more general, all-inclusive list in the first few papers you read. Let me know if you find a primer set that seems interesting and I can forward you any papers I may have on that specific primer set.
You can just order species specific primers for PRC. Or you can buy universal primer for bacteria to perform amplification. Then you can send samples to sequence analysis.
You can use universal Primers that target the v3/v4 regions and followed by Miseq sequencing. targetting the v3-v4 regions allow you to get a greater depth to differentiate at the species level between bacteria
Got it, Thanks for your highly appreciated comments and suggestions Maurice Ekpenyong Karen A. Darbinyan Marwan E Majzoub
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