Detection and quantification of nonhuman species

Forensic analysis of non-human evidence is gaining importance and becoming widely used in forensic laboratories for the identification of animal material recovered from the crime scene (usually pet hairs). A SYBR Green quantitative real-time PCR assay has been developed for the quantification of genomic DNA extracted from domestic cat samples (Menotti-Raymond et al., 2003)

Investigation of the illegal trade in endangered species is another field of application of real-time quantitative PCR. A highly sensitive tiger-specific realtime PCR assay has been described using primers specific to the tiger mitochon-drial cytochrome b gene. Successful amplification has been demonstrated from blood, hair and bone as well as from a range of traditional Chinese medicines spiked with 0.5% tiger bone (Wetton et al., 2004).

Real-time quantitative PCR is also an emerging technique in ancient DNA studies of non-human species. Poinar et al. (2003) quantitated the number of mitochondrial 16S rDNA copies for fragments of three different lengths (114/252/522 bp) by using a TaqMan real-time PCR assay from ancient copro-lite remains, demonstrating that there was roughly a 100-fold drop in the number of amplifiable mtDNA molecules for every doubling in amplification length.

Quantitative PCR analysis of DNA from non-invasive samples with low DNA content is also a useful tool for molecular characterization of wild animal populations in molecular ecology, including nuDNA quantification (Morin et al., 2001) and sex determination (Morin et al., 2005).

Real-time quantitative PCR is also a rapid and highly sensitive methodological tool for detection, identification and individualization of microbial agents that could be used in bioterrorist acts. For instance, real-time PCR assays are routinely used to detect the presence of DNA from Bacillus anthracis in environmental samples by using both unique plasmid-borne and chromosomal genes (Jones et al., 2005). Other highly sensitive real-time PCR assays for the detection of potential bioterrorism agents such as Yersinia Pestis, Francisella tularensis, Brucella spp. and Burkholderia spp. have also been developed (see a review of the assays for biodefence used at the USA Armed Forces Institute of Pathology in Jones et al., 2005).

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