The importance of an internal amplification control for diagnostic qPCR assay

It is still surprising that nowadays, a major drawback of most published PCRs, is that they do not contain a proper Internal Amplification Control (IAC). An IAC is a non-target DNA sequence present in the very same sample tube, which is co-amplified simultaneously with the target sequence. In order to understand the use of optimal IAC, the review performed by Hoorfar and co-workers should be mandatory reading for all qPCR makers (Hoorfar et al., 2004). In a PCR with an IAC, a control signal will always be produced when there is no target sequence present. When neither IAC signal nor target signal is produced, the PCR has failed. His study presented several recommendations for optimal IAC to be used in diagnostic PCR which are really interesting: Target DNA and IAC should share the same primer-binding sites. IAC amplicons should be easily distinguishable from the target DNA amplicons. It is not strictly necessary that the amplification efficiencies of the target and IAC DNA are identical. The source of IAC should be plasmid DNA carrying the cloned IAC sequence or purified PCR products. Depending of the detection method of the assay, the IAC should be detected by sequence-dependent hybridization probes or by size after gel electrophoresis. Only high purified templates should be used. The concentration of the IAC should be determined by titration and its amount in the assay should be as low as possible, while still eliciting a signal through amplification. IAC should be added to the PCR mix to ensure equal distribution in all PCR tubes. Sometimes, however it is necessary to amplify a sequence of a “housekeeping” gene (16S ribosomal gen, i.e.) in order to check the integrity of the purified DNA, but inclusion of an IAC should not be replaced. Also, inclusion of an IAC should be required in any PCR developed for diagnostic use, either as a detection or subtyping tool. BIBLIOGRAPHY Written by Ana Camacho.