I am developing a dPCR assay to detect genomic DNA. it is my understanding that we can calculate LOB from the replicate values of NTC and use that to determine LOD in your assay, but does anyone aware of any publication that discussed setting up limits of positive signals in NTC or negative samples in terms of copies per uL in dPCR platform? Thanks in advice for your input.
Hi,
In general, you should aim for 0 positives in your NTC. A good assay should not have any background.
More important then an NTC, is - if applicable - a true negative control (NC or process NC) (for example a WT if you want to quantify mutants). That said, the number of positives in an NTC/NC that you can tolerate, will indeed depend on the LOD you want to achieve.
Before tackling your question, it is important to clarify these definitions:
LOB = highest result that is likely to be observed in a blank sample
LOD = lowest concentration at which the analyte can be detected with a given certainty (95%)
You can indeed estimate your LOD, based on your LOD (blanks) + your low concentration sample. Formulas are nicely described in this (qPCR) publication: Article Methods to determine limit of detection and limit of quantif...
In terms of guidelines, you have for example the ISO20395, dMIQE and CLSI guidelines that might help you further.
Hi, there was a typo in my answer. It should have been: You can indeed estimate your LOD, based on your LOB (blanks) + your low concentration sample.