A vertebrate-specific qPCR assay as an endogenous internal control for robust species identification

The identification of vertebrate species is important in numerous fields such as ecology, archaeology, and food and forensic sciences. Real-time quantitative PCR (qPCR) assays specific for one vertebrate species are promising approaches for species identification, although there are several drawbacks such as difficulty determining whether the detected DNA is authentic or a contaminant. Here, we describe a qPCR assay specific for vertebrate mitochondrial DNA (mtDNA) which can overcome these drawbacks. Since we found that mitochondrial 16S rRNA contains regions that are perfectly (not highly) conserved across virtually all vertebrates, but are variable in invertebrates, we were able to design a vertebrate-specific qPCR assay by placing primers/probe within these regions. The specificity and accuracy of this assay were validated with representative vertebrate and invertebrate samples. This assay detected DNA from all vertebrate samples, but not from any invertebrate samples. In addition, this assay was able to quantify vertebrate mtDNAs as accurately as previously reported species-specific qPCR assays. The results demonstrated it is feasible to quantify vertebrate mtDNA specifically and accurately in a sample. This means that it is possible to determine the ratio of specific vertebrate species mtDNA to total vertebrate mtDNA in a sample. In conjunction with this assay as an endogenous internal control, species-specific qPCR assays will allow for the robust identification of vertebrate species.