We are currently trying to identify presence of babesia bovis, babesia bigemina, and anaplasma marginale in DNA isolated from white blood cells. However, the amplification plot values start approximately in cycle 5 and do not show exponential growth followed by a plateau, as other Real Time PCR reactiones we have done in the past, which usually start in cycle 20 or above and show the aforementioned amplification plot characteristics. Is this graph and CT enough to validate the presence of such organisms in our samples?
You won't be able to use those results quantitatively (or even arguably qualitatively). Can you show us the amplification plots of a NTC signal and a sample of your choice? As it stands, it is very hard to separate noise from signals based on the pictures. Although i am not a microbe expert, typically PCR results tend to be >10 Cts.
Article Use of a Real Time PCR for detecting subspecies of Babesia canis
firstly check the DNA quality then try to standrdize with positive control.
There's something wrong with the PCR. Only the dark blue(?) curve in IMG_3822.JPG shows a shape, more or less typical for amplification. For me, the curves look pretty much like background. Only by speculation, maybe you have an inhibitory effect by an excess of human DNA over your target DNA.
You need to include a negative (no template control, NTC) to see the baseline and at least one good positive control for each target to see amplification. I would suggest using a well characterized positive control in different dilution steps (DNA concentrations, if DNA content is known) to figure out, how the PCR works with different amounts of target. At a certain (high!) target concentration, you end up with inhibition. Same holds true if you have a high imbalance between your target DNA and human DNA (might be the problem here). To figure that out, you could do spiking experiments with positive control spiked in diluted white blood cell DNA preparations.
Based on experience, strong positive samples (that is for example HIV with 100 millions copy per ml) may show a CT between 12 and 15 (in commercial assays). With Babesia/Anaplasma from blood I would expect CT >20 at best.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics