Mitochondrial DNA digital PCR improves metastasis quantification in the chick chorioallantoic membrane (CAM) assay.

The chick chorioallantoic membrane (CAM) assay is an in vivo metastasis model, yet traditional Alu-qPCR quantification suffers from non-specific background noise in avian tissue. To improve analytical specificity, we developed a species-specific digital PCR (dPCR) assay targeting the human mitochondrial locus MT-ND4. While Alu-qPCR generated false positives in chicken-only DNA, MT-ND4 dPCR provided high species specificity and robust relative quantification of human mitochondrial DNA across various embryonic organs. As a proof of concept in five embryos, organ-dependent differences in metastatic burden were detected (p < 0.001) with high technical precision. This MT-ND4 dPCR assay avoids false-positive overestimations, providing a specific tool for quantifying human metastatic burden in the CAM model.