Use of a universal hydroxymethylbilane synthase (HMBS)-based PCR as an endogenous internal control and to enable typing of mammalian DNAs

There is a need for an endogenous internal control (EIC) for PCRs to monitor the quality and quantity of DNA in test samples. We designed and validated a fluorescence resonance energy transfer (FRET)-PCR targeting the mammalian homolog of the hydroxymethylbilane synthase (HMBS) gene as an EIC for PCRs on mammals. The designed FRET-PCR detected the HMBS gene in whole blood of 13 mammalian species collected from eight countries and in 11 murine organs/tissues. It could also be used to quantify the volumes of mammalian blood meals in mosquitoes and by sequencing the amplicons obtained we could determine the mammalian species (6) from which the meal was obtained. The FRET-PCR proved highly sensitive (one gene copy in 0.05 ng tissue or 0.5 nl whole blood) and specific with no false negative or positive results. The high sensitivity and specificity of the FRET-PCR and its ability to differentiate mammalian species makes it an ideal EIC for PCRs involving mammals and a useful tool for hematophagous insect studies.