Forensic hair analysis to identify animal species on a case of pet animal abuse.

As part of an investigation of a case of pet animal abuse, we attempted to identify small mammalian species by morphological analysis and single-nucleotide polymorphism (SNP) typing of the cytochrome b gene using guard hairs as an analytical material. Guard hair samples from several species were measured for length, width, medulla formation, and cuticle scale pattern under a light microscope or scanning electron microscope. These samples were also analyzed for SNPs in the cytochrome b gene using a multiplex single-base primer extension reaction. Morphological analysis of cuticle scale pattern and medulla formation was able to discriminate ferret hairs from other hair samples that included rabbit, gerbil, degu, and Djungarian hamster. However, this also revealed a similarity of the guard hairs of the ferret (Mustela putorius furo) and Japanese weasel (Mustela itatsi). Although at three sites, the nucleotide color signals of SNPs in the cytochrome b gene could be used to discriminate completely among human, dog, and gerbil, the signals for cat, ferret, and Japanese weasel occurred at the same nucleotide sites. Unfortunately, no signals were obtained from degu, Djungarian hamster, and rabbit hairs. Although the discriminated hair samples were 100% identical to those of the ferret, there was only a 5% difference from Japanese weasel in the partial sequence of the cytochrome b gene. Construction of a database of mammalian hairs would be useful not only in forensic science, but also for investigating smuggling of endangered species in contravention of the Washington Convention.

Study of animal species (human, dog and cat) identification using a multiplex single-base primer extension reaction in the cytochrome b gene.

We developed a simple method for animal species identification of humans, dogs and cats, using a multiplex single-base primer extension reaction in the cytochrome b gene. Using this method, three points of a single nucleotide in the cytochrome b gene were examined in these species using primers of different lengths. Our method was found to be able to successfully identify humans (26 samples), dogs (21 samples) and cats (9 samples), and no differences were found among the samples from each animal species in this study. The amount of template DNA required was over 0.01 ng for humans and dogs, and over 0.1 ng for cats. The present method was able to identify animal species from hair shaft (2 cm) and forensic casework samples (blood stains and hair shafts), and is thus a useful tool for animal species (human, dog and cat) identification in forensic science.

Applicability of Nanotrap Sg as a semen detection kit before male-specific DNA profiling in sexual assaults.

A commercially available semen detection kit, Nanotrap Sg, which employs a one-step detection test based on immunochromatographic assay for the semenogelin protein, was evaluated for profiling male-specific DNA in sexual assault casework samples. While semen diluted with phosphate-buffered saline held and kept at 4 degrees C for 1 week showed a relatively strong signal intensity with Nanotrap Sg, the signal intensity was decreased by dilution after storage at 4 degrees C or freezing and thawing repeated more than three times. The reproducibility of Nanotrap Sg was tested on a total of 174 sexual assault casework samples from three forensic laboratories using intra- and interassay and no variation was observed in the semenogelin (Sg) signal. The positive signal ratio was 12.6% higher for prostate-specific antigen immunochromatographic membrane tests than Nanotrap Sg. Although spermatozoa were not confirmed in 61 (35%) out of 174 samples, Sg-positive signals could be detected from 41 (67%) of the 61 samples. Female genetic profiles could be observed in 95% of the samples, which tested negative for Sg on the Nanotrap Sg test, but no male genetic profiles could be observed. These results suggest that Nanotrap Sg can positively identify samples containing male DNA even in the absence of detectable intact spermatozoa. Further, Sg-positive signals identified samples for which male-specific DNA profiling could be performed, even if no sperm could be detected from the sample. The potential of Nanotrap Sg for identifying forensic samples with male-specific DNA was clearly demonstrated.