Development and validation of simultaneous identification of 26 mammalian and poultry species by a multiplex assay.

A multiplex PCR assay was developed to simultaneously identify 22 mammalian species (alpaca, Asiatic black bear, Bactrian camel, brown rat, cat, cattle, common raccoon, dog, European rabbit, goat, horse, house mouse, human, Japanese badger, Japanese wild boar, masked palm civet, pig, raccoon dog, red fox, sheep, Siberian weasel, and sika deer) and four poultry species (chicken, domestic turkey, Japanese quail, and mallard), even from a biological sample containing a DNA mixture of multiple species. The assay was designed to identify species through multiplex PCR and capillary electrophoresis, with a combination of amplification of a partial region of the mitochondrial D-loop by universal primer sets and a partial region of the cytochrome b (cyt b) gene by species-specific primer sets. The assay was highly sensitive, with a detection limit of 100 copies of mitochondrial DNA. The assay's ability to identify species from complex DNA mixtures was demonstrated using an experimental sample consisting of 10 species. Efficacy, accuracy, and reliability of the assay were validated for use in forensic analysis with the guidelines of Scientific Working Group on DNA Analysis Methods (SWGDAM). The multiplex PCR assay developed in this study enables cost-effective, highly sensitive, and simultaneous species identification without massively parallel sequencing (MPS) platforms. Thus, the technique described is straightforward and suitable for routine forensic investigations.

Current issues for mammalian species identification in forensic science: a review.

Mammalian species identification is one of the important issues in forensic science. Determining the origins of non-human biological material found at crime scenes can increase the possibility of identifying the true culprit by narrowing down the range of suspects. Although many techniques based on mitochondrial DNA (mtDNA) have been developed, challenges remain to cost-effectively identify species from degraded samples containing a mixture of DNA from multiple species and to standardize procedures for mammalian species identification. This review evaluates the reliability and versatility of mtDNA-based techniques to reveal obstacles to the establishment of standard analytical methods, with a particular focus on DNA mixtures. When samples contain a mixture of DNA from multiple species, the interpretation of sequencing analysis results is difficult. Although DNA metabarcoding using next-generation sequencing (NGS) technologies can overcome the DNA mixture problem, DNA metabarcoding is not suitable for the type of small-scale analysis routinely performed by local forensic laboratories, primarily because it is costly and time-consuming. By contrast, fluorescent multiplex PCR analysis enables cost-effective and simultaneous species identification from suboptimal samples, although the number of identifiable species is currently limited in comparison with sequencing techniques. The advantages and limitations of current techniques presented in this review indicate that multiplex PCR analysis will continue to be important for mammalian species identification in forensic casework analysis. Further developments in multiplex PCR analysis that enable the identification of an increased number of species will play a key step for standardization efforts among forensic laboratories.

Involvement of soil bacteria in ABO blood mistyping.

The current study investigated whether ABO blood mistyping of human biological samples is induced by soil bacteria. A total of 380 bacterial strains were isolated from 50 discrete soil samples using human blood agar, and glycosidase activity evaluated for all strains using 4-nitropheny glycosides (4-nitrophenyl n-acetyl-α-D-galactosaminide, 4-nitrophenyl-α-D-galactopyranoside, 4-nitrophenyl-α-L-fucopyranoside) as substrates. Thirteen strains possessed α-galactosidase activity, and 16S rRNA sequence analysis revealed a close relatedness to the genus Bacillus. An indirect competitive enzyme-linked immunosorbent assay confirmed seven strains exhibited type B antigen degradation activity. These results demonstrated that 1.8% of the bacteria isolated from soil, were Bacillus sp., possessed galactosidase activity, and had the potential to cause ABO blood mistyping.

Development of an indirect competitive ELISA for the detection of ABO blood group antigens.

We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of ABO blood group antigens in human samples; in particular for blood stains. ABO blood group antigens conjugated to polyacrylamide were used for immobilized antigen. ABO blood group antigens were extracted from blood stains using a novel method involving pre-incubation with proteinase K (PK), followed by heat treatment. The extracts (analytes) were combined with either anti-A or -B monoclonal antibodies (mAbs), and added directly to the antigen-coated wells. The anti-A and -B mAbs were captured by either ABO blood group antigens present in the analyte or by the immobilized blood group antigens. Peroxidase-conjugated anti-mouse IgM antibody was used to detect anti-A and -B mAbs complexed with immobilized blood group antigens, and a colorimetric reaction using o-phenylenediamine/H2O2 used for its measurement. The ELISA developed in this study was able to detect blood group antigens in blood, saliva and blood stains. The detection limit for unknown blood, saliva and blood stain were determined as 1:200, 1:32 and 1:16. Overall the ABO blood grouping ELISA can be used with relative ease for the high throughput screening of biological samples for the detection of ABO blood group antigens.

Methanothermobacter tenebrarum sp. nov., a hydrogenotrophic, thermophilic methanogen isolated from gas-associated formation water of a natural gas field.

A thermophilic and hydrogenotrophic methanogen, strain RMAS(T), was isolated from gas-associated formation water of a gas-producing well in a natural gas field in Japan. Strain RMAS(T) grew solely on H(2)/CO(2) but required Casamino acids, tryptone, yeast extract or vitamins for growth. Growth of strain RMAS(T) was stimulated by acetate. Cells were non-motile, straight rods (0.5×3.5-10.5 µm) and occurred singly or in pairs. Bundles of fimbriae occurred at both poles of cells and the cell wall was thick (approximately 21 nm, as revealed by ultrathin section electron microscopy). Strain RMAS(T) grew at 45-80 °C (optimum, 70 °C), at pH 5.8-8.7 (optimum, pH 6.9-7.7) and with 0.001-20 g NaCl l(-1) (optimum, 2.5 g NaCl l(-1)). Phylogenetic analysis revealed that Methanothermobacter thermautotrophicus ΔH(T) was most closely related to the isolate (95.7 % 16S rRNA gene sequence similarity). On the basis of morphological, phenotypic and phylogenetic characteristics, it is clear that strain RMAS(T) represents a novel species of the genus Methanothermobacter, for which we propose the name Methanothermobacter tenebrarum sp. nov. The type strain is RMAS(T) ( = DSM 23052(T) = JCM 16532(T) = NBRC 106236(T)).