During specificity test of a diagnostic, when it challenged against plasmid DNA from a non-target species at a very high concentrations of DNA non-target (10E8 copies/ul) there was amplification. However, at lower concentrations this did not occur. Non-specific amplification may have occurred due to stochastic events, ie, since all the components necessary for amplification to occur occur (except for the target DNA), the physical interaction between the molecules could have favored non-specific amplification, considering this extreme situation of High concentration of non-target DNA?
In short, yes, it can happen.
In real-time PCR assays that are designed to be very sensitive you can sometimes have amplification of a nonspecific target. There are several possibilities. Real-time PCR assays, although often more specific than conventional PCR assays because of additional specificity provided by the probe. However, some assays will tolerate one or more mismatches in the probe and/or primer sequence. One possibity, then is amplification of a phylogenetically related organism that has a similar sequence.
To find out whether you have nonspecific amplification of a single template, I would recommend sequencing the PCR product as a first step. Also, if you find that you are amplifying a nonspecific target, you can redesign the assay to exclude that nonspecific target and you can sometimes slightly improve specificity by adjusting the cycling parameters, such as by slightly increasing the annealing temperature to improve specificity, depending on the annealing temperature of your primers and probe.
Best regards,
Marcus
Marcus Crim,
First of all, thanks for your help.
About your suggestions, I already done that you said. However, un specific amplification happen just when I'm using a very high concentration of plasmid DNA from a closely related species to the target. In fact, there are just four divergent nucleotides into the hibridization site of the probe between those two species, but teorically, this condition would not enough to amplificiation occur. Curiosily, when I challenge the probe using genomic DNA or lower concentrations of plasmid DNA from this closely related species, there was no amplification.
Do you know articles that address these strange results in qPCR in situations of high concentrations of a DNA background?
Best wishes.
Dalila.
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