Hi everyone
Mycoplasma detection has been a major challenge for me lately. I do cell culture and use qPCR to detect mycoplasma. In addition to controls(+,-) and NTC, I use 3 different strains of mycoplasma as standards (M. orale, M. pneumoniae and M. fermentans).
Some Cts relating to standards are extremely high (sometimes 45!) for a few weeks.
(This is while I count the standard samples with a flow cytometer before DNA-Extraction and at least 13x10^6 cells are counted in it.)
The samples and wells in which Ct indicates greater than predicted (I consider ≤35 to be an acceptable Ct and I had before) appear to be entirely random.
I have tested different modes, and the results are uninterpretable. We have two PCR devices, and the problem is the same in both.
All samples, controls and standards are pipetted into two wells (double determination).
Does anyone have any ideas?
We had a similar situation due to contamination of solutions from DNA extraction kits.
In an early infection, with a very low viral level, it is possible that the test cannot detect the infection; in such cases, the test can result in a false-negative result. Additionally, a pre-analytical problem can result in a false-negative result. The best example is poor specimen collection
Dhuha Abdullah Kadhim I use the Sartorius(Mycoplasma detection) kit.
The strange thing is that this kit used to work better with the approved method (GMP) and for some time the number of unexplained errors became very high.
I am looking for an idea to find the way to reduce errors. I've made some changes (temperature cycling, increase in fluid containing mycoplasma DNA...) and the results are not explainable!!!
1. The problem might be in a DNA extraction, during the process you are loosing some DNA. Try another kit.
2. Remember that qPCR detects DNA present in a sample while flow cytometry detects cells. You can have much more copies of DNA eg. from dead/destroyed cells than cells detected in cytometry.
Thank you for your suggestion. As you said, we count the number of cells and this amount is much more than what is required for a reliable PCR.
We do it like this:
1- Cells infected with the desired Mycoplasma are purchased.
2- We count the number of cells.
3- Gene extraction
Important point:
4- We do a PCR test for the gene of the standard samples (whose gene has been extracted). If they (Standards) show CT less than 35, we use them in detection.
I thought maybe we don't have proper maintenance conditions. We put the extracted genes in a -20 freezer.
On the other hand, the controls (+,-,NTC) of the used kit (Sartorius) all respond within the reliable range.
On the other hand, the samples are used in two wells. I mean, we have 3 standards, each of which we use twice. A total of 6 wells are filled with 3 standards. But the result is strange. For example, one CT well shows less than 35 and another well that has the same standard shows more than 35 (in some cases even 45).
And more importantly, every time 2 or 3 CT standards are unacceptable from the total of 6 standards (2x3), and it may be any of them (that is, one well from one standard, or 2 wells from the same standard, or 2 wells from 2 different standards, etc. ...)
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Materials Chemistry
- Polymer Chemistry