I would like to quantify Escherichia coli K-12 by qPCR, discriminating it from other coli and bacteria. I have found this primer set on
DOI: 10.1128/aem.61.11.4135-4139.1995
but on my hands it did not work. I used boiled DNA (1 mL bacterial suspension in culture medium heated at 99 degrees for 10 mins), with an annealing at 60 degrees as reported in the paper but I got no amplification.
Is there a better PCR assay to specifically detect K-12?
Thank you
Did you have any positive controls for your PCR reaction and primers (like a known K12 strain) and also control primers for your template. You need to be sure the assay actually works first before worrying about the correct primers.
Also did you really use 1ml of a bacterial suspension? How much of that went into your PCR reaction? That sounds like a lot of bacteria and bacterial debris is often inhibitory to PCR reactions which might be the problem.
I completely agree, but I never ran this assay so I know nothing about it. The main problem is that I don't have a true positive control. I have used a K-12 strain (or so I am told) and some derivatives such as HB101 and DH5. I added 10 uL to a 25 uL total volume but I agree that the debris might be an inhibitor. Shall I stick to extracting the DNA? Shall I order a synthetic oligo containing the primer sequences to have a sure control? Is boiled DNA a good working option? (for sure is cheap).
Either of those strains (HB101 or DH5) are suitable positive control strains so if those aren’t working then you have a different problem. I think you are adding too much template, I would scale back to 1ul in 25ul reaction, or even less.
But if you only have a few strains to test then you might consider doing a real DNA extraction, but try the reduced template first.
I tried tweaking the load as you suggested. In the gel above, I have K12 and HB101 in tandem, with 10, 5, and 1 µL input, first with boiled DNA then with DNA extracts (and one NTC to separate them). I used too little marker, but the main outcome is that there is single band. It is clear that DNA extraction always works whereas 1 µL is amplified. Hence, as you pointed out, too much lysate inhibits the reaction. Case solved, thank you!
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