Tomoaki Mitani

Identification of animal species using the partial sequences in the mitochondrial 16S rRNA gene

This study investigated a PCR direct sequencing method for species identification by analyzing partial sequences in the mitochondrial 16S rRNA genes of many animal species amplified with universal primers. Samples from 182 vertebrates and 103 invertebrates were analyzed, and the sequences could be obtained in 182 and 72 species, respectively. The sequence divergence was sufficient to identify the species at the level of genus with the aid of the GenBank database and the BLAST tool. This method could be a powerful tool for animal species identification, especially in forensic cases in which many unknown biological samples should be analyzed.

Evaluation of prostate-specific antigen (PSA) membrane test for forensic examination of semen.

Prostate-specific antigen (PSA) is a glycoprotein produced by the prostatic gland and functions to liquefy the seminal fluid. Recently several PSA membrane test kits utilizing an immunochromatographic assay are commercially available for clinical screening of PSA concentration in serum. In this study, we applied the three PSA test kits to forensic examination of semen, and evaluated the sensitivity and specificity of the PSA test kits. The specificity were the same, but SMITEST PSA Card had the highest sensitivity among the three kits. Furthermore we certified that the SMITEST PSA Card was little affected by heat or contaminants such as other body fluids, reagent of preliminary test for semen stain and spermicides, and the PSA kit was applied to 30 casework samples.

Se genotyping following allele-specific polymerase chain reaction amplification

The polymorphism of the Sec2 gene, which determines Se blood type, has been reported. This study presents an Se genotyping system by the allele-specific polymerase chain reaction amplification method. The Se, sej and se(fus) alleles were amplified using allele-specific primers. The Sec1, Sec2 and se(fus) genes were analyzed by DNA sequencing. The 299-bp Se, 146-bp sej and/or 312-bp se(fus) allele-specific products were amplified and detected in the native polyacrylamide gel. The 314th-316th nucleotides of the Sec1 gene were CCC, which were different from the nucleotides GGG reported previously by Kelly et al. [J Biol Chem 270 (1995) 4640]. This Se genotyping system is a simple method available for the forensic science field in Japan. The crossover region of the se(fus) gene is a 164-bp stretch corresponding to the regions between the 253rd and 416th of the Sec1 gene and between the 211th and 374th of the Sec2 gene.

A case of DNA analysis of ABO blood group variant allele A2

The ABO phenotype of a bloodstain (B) on a knife that was used as a weapon in an attempted murder case was found to be different from that of the Peruvian victims blood (AB). Serological analysis showed that the A-antigenicity was much weaker than B antigenicity, suggesting that the victims phenotype was A(2)B or A(3)B. So, the ABO genotypes of the knife bloodstain and the victims blood were determined by DNA analysis. The 261st G deletion, specific to the O(1) allele, was not detected in the specimens by restriction fragment length polymorphism analysis. Also, the 871st A, specific to the A(3) allele, was not found by the allele-specific amplification method. Amplified product length polymorphism and direct sequencing methods finally demonstrated that the typical B sequence was found in one allele and a single C deletion in the 1,059th-1,061st C stretch in the other allele, indicating that the ABO phenotype of the bloodstain and victims blood were A(2)B.

Effects of solvent displacement on sensitivity and specificity of monoclonal antibodies for ABO blood grouping of forensic specimens with an absorption-elution test.

Using commercially-available monoclonal antibodies (Bioclone, Neo Kokusai, Monoclonal Wako, Gamma Clone and Seraclone), ABO blood grouping of forensic specimens such as bloodstains, salivary stains, seminal stains, nails, hair and cerebral dura mater was performed with an absorption-elution test. Salivary stains, seminal stains and nails were not typed correctly using the antibodies other than Bioclone reagents, while precise grouping of bloodstains was performed using most antibodies. When hair and dura mater were tested, all of the antibodies induced weak or non-specific haemagglutination, hence correct grouping was not achieved. When the antibody solvents were displaced with 5-20% bovine serum albumin in saline, human serum of the group AB donor, or serum of chicken, sheep or bovine, titers of the reagents increased 2-8 times. Hair and dura mater were able to be typed using Bioclone reagents after solvent displacement with human AB or sheep serum, whereas displacement with the other solvents enhanced non-specific reactions.

Examination of seminal stain by HPLC assay of phenolphthalein

Quantitative high performance liquid chromatography (HPLC) to detect semen was investigated in this study. Briefly, 1cm of a gauze thread with a seminal stain was soaked in the reaction mixture (phenolphthalein diphosphate tetrasodium dissolved in acetate buffer) for 5-10 min, and the supernatant was analyzed by HPLC with a spectrophotometric detector. Phenolphthalein was liberated from the reagent in the presence of acid phosphatase, and the liberated phenolphthalein was detected objectively and was unaffected by blood contamination. Since liberation of phenolphthalein from the reagent occurred slightly in control negative samples, the cut-off value of the examination should be set at 1.0 microg/ml.

The proof of flat-line scalp EEGs of brain dead patients by an automatic EEG analysis system

Scalp electroencephalograms (EEGs) of brain dead patients are macroscopically flat under 7 or 10 microV/mm electroencephalograph sensitivity, but significant noises are detected in EEGs under 2 microV/mm sensitivity, interfering with the analysis. EEGs of 20 brain dead patients (17-76 years old) were therefore analyzed quantitatively as equivalent electric potentials in frequency bands delta, theta, alpha and beta using the automatic EEG analysis system developed in Kansai Medical University. The equivalent electric potentials in each band were about or less than 1 microV, which is used as a criterion of judgment of flat-line EEGs or brain death. Then, macroscopically flat EEGs of 12 comatose patients including infants (3-67 years old) were analyzed by the system, confirming their brain death. Thus, the automatic EEG analysis system could be used as a supporting tool to confirm flat-line EEGs of brain dead patients. ATAMAP II for Windows software was also evaluated.