Hi everyone. I'm diagnosing SARS-CoV-2 using LightMix Modular SARS-CoV (COVID19) E-gene. However, there was a case where a sample showed a curve with cq = 35 (see photos) and was reported as positive. The next day the sample was taken again and the result was without amplification. There are several possibilities that I contemplate:
1. Cross contamination. However, I consider this unlikely because there were only 3 positive samples out of 94 not including negative control (only with internal control). The positives are not contiguous.
2. The patient was finishing the infectious process and therefore was undetectable the next day. No symptoms were reported.
3. Something wrong with the probe. But here I am not sure if it is possible for non-specific amplification. The behavior of the curve is not perfect, but there seems to be a true amplification. My question is about this possibility.
The green curve is the internal control (exogenous). The red is for SARS-CoV-2 (FAM).
J. Andrés Narváez-Pardo Did you try to run the sample in agarose gel?
...hard to say. To exclude problems with the probe (what is not likely in my opinion) you can run an agarose gel as suggested by Giorgia. I would also recommend to repeat this sample and the next day sample from the same patient. I think the most likely explanation is a very low level of the target thus the results being affected by stochastic fluctuations (Poisson distribution).
Best,
Norman
Thanks, Giorgia. It’s a good idea, but this is a clinic laboratory and there’s no time to run a gel. More than 1400 samples are processed daily.
J. Andrés Narváez-Pardo analyse by gel only a sample that run godd and two three that have a strange amplification curve only to check what happens.
Dod you try a run with different contration of primer probe and cDNA?