The performance of the real-time immuno-PCR assay can be tested by running a number of control reactions in parallel with the test samples. The background signal from non-specifically bound assay components is assessed by a background control (BC) that contains the same amount of antibodies and DNA as positive samples, but without antigen. The BC signal is caused by non-specific adsorption of reaction components and should have as high Ct as possible. The second control is the no template control (NTC). The NTC contains the real-time PCR mastermix only. It reveals any contamination of the mastermix and its Ct reflects primer-dimer formation. If the NTC and BC have similar Ct, primer-dimer formation limits sensitivity, and it is probably a good idea to redesign the real-time PCR assay.

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