I am trying to do a multiplex PCR to detect two bacterial species using the ABI 7300 real time PCR system. So far the primers for these species and their Taqman probes have been optimised but I still need to find a primer set to be used as control to make sure that the bacterial DNA extraction has been successful. I have tried two different primer/probe sets as well as a set of LUX primers but none have worked properly. Do you have any idea which gene can be targeted for the real time PCR to work efficiently?
Hi,
you could use a generic 16S Primer pair which would detect all possible bacterial species. Let me know if you would like to get more infos and support as I am currently working on something similar and look for field testers
Sven
@ Sven Reister Thank you for your answer!
I have used two different primer sets and they work well in end-point PCR but not real-time due to the Taqman probe, so i was wondering if there was something else I could try.
Hi,
As Swen mentioned, 16S rRNA should be a good control but if that doesn't work for you, the below paper mentions a list of genes(rpoA,gyrB,gap,rho etc) that have been experimentally validated as reference genes. You can have a look.
Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis
https://link.springer.com/article/10.1007%2Fs10482-015-0524-1
Even references are mentioned, so you can look for studies which used Taqman probe for RT qPCR and go with that gene.
All the best!
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