Biomedical subjects

Hiroaki Nakanishi

Publications and source records attributed to Hiroaki Nakanishi.

5 recordsLinked to original sources

PubMed2026

Development of a universal primer set for the identification of enterovirus serotypes using next-generation sequencing.

Enteroviruses have been recognized as major etiological agents of viral myocarditis, a significant contributor to sudden cardiac death. However, the causative virus genomes are rarely identified in forensic autopsy specimens, because extensive nucleic acid degradation compromises analysis. The aim of this study was to develop a next-generation sequencing (NGS)-based method for detecting and identifying the serotype of enteroviruses, which are one of the primary viral causes of myocarditis. We developed a primer set using sequences from 21 representative enterovirus serotypes. The sensitivity of the method was assessed using 13 artificial enteroviral DNA constructs. To confirm the clinical performance of our proposed method, a subset of nasal specimens was analyzed independently by a commercial laboratory using the BioFire Respiratory Panel 2.1 microarray-based viral screening assay, and the results were compared with those obtained using our method. Our method was able to detect as few as 10 copies of artificial DNA and successfully identified all 13 evaluated serotypes, even within a mixture sample. Compared with the BioFire Respiratory Panel 2.1 microarray-based assay, our NGS-based method demonstrated concordant results in 12 out of 14 evaluated cases, showing comparable detection sensitivity. In a trial application of the method, we were also able to detect and serotype enteroviruses in frozen myocardial samples from three forensic myocarditis cases. Overall, the findings of this study suggest that the NGS-based method developed here is a promising diagnostic approach for identifying the causative pathogens of viral myocarditis in forensic autopsy cases.

Autopsy myocardial sample↗

PubMed2006

Preparation of latex reagents combined with IgM and its F(ab')2 fragment from commercial ABO blood grouping reagent.

To achieve a rapid assay for ABO blood grouping using a latex reagent, two latex reagents were produced, one of which combined with mouse monoclonal immunoglobulin M (IgM) isolated from commercial ABO blood grouping reagent, and the other of which combined with its F(ab')2 fragment prepared by cold pepsin digestion. The latex reagent adsorbing the F(ab')2 fragment was able to detect the 1000-fold diluted saliva and provided much better sensitivity than that of IgM. This suggests that the difference in sensitivity between the two latex reagents is responsible for adsorption orientation of the antigen site on the latex particles. The new assay successfully completed the ABO blood grouping of cigarette ends within 30 min.

ABO Blood-Group System↗

PubMed2006

Real-time monitoring of antibody secretion from hybridomas on a microchip by time-resolved luminescence anisotropy analysis.

This article presents a real-time monitoring system for cellular analysis using micro total analysis systems technology. Time-resolved luminescence anisotropy analysis was adopted for real-time detection of small amounts of a target protein produced by a small number of cells. The system was tested by real-time monitoring of the antibody secretion by hybridomas. The cells were successfully cultivated in a micro-incubation chamber (240 nl) fabricated on a microchip. The quantification of the antibody was achieved using the Ru(II) complex-labeled Staphylococcus aureus protein A probe, which can bind specifically to the Fc region of the antibody. Using this system, we detected as little as 24 fmol of immunoglobulin G under physiological conditions without the bound/free separation protocol. We successfully achieved real-time and quantitative monitoring of small amounts of antibody production by approximately 200 hybridoma cells. This method could be applied to various cellular analyses using small numbers of cells.

Animals↗

PubMed2004

Separation of long DNA molecules by quartz nanopillar chips under a direct current electric field.

We have established the nanofabrication technique for constructing nanopillars with high aspect ratio (100-500 nm diameter and 500-5000 nm tall) inside a microchannel on a quartz chip. The size of pillars and the spacing between pillars are designed as a DNA sieving matrix for optimal analysis of large DNA fragments over a few kilobase pairs (kbp). A chip with nanopillar channel and simple cross injector was developed based on the optimal design and applied to the separation of DNA fragments (1-38 kbp) and large DNA fragments (lambda DNA, 48.5 kbp; T4 DNA, 165.6 kbp) that are difficult to separate on conventional gel electrophoresis and capillary electrophoresis without a pulsed-field technique. DNA fragments ranging from 1 to 38 kbp were separated as clear bands, and furthermore, the mixture of lambda DNA and T4 DNA was successfully separated by a 380-microm-long nanopillar channel within only 10 s even under a direct current (dc) electric field. Theoretical plate number N of the channel (380-1450 microm long) was 1000-3000 (0.7 x 10(6)-2.1 x 10(6) plates/m). A single DNA molecule observation during electrophoresis in a nanopillar channel revealed that the optimal nanopillars induced T4 DNA to form a narrow U-shaped conformation during electrophoresis whereas lambda DNA kept a rather spherical conformation. We demonstrated that, even under a dc electric field, the optimal nanopillar dimensions depend on a gyration radius of DNA molecule that made it possible to separate large DNA fragments in a short time.

DNA↗

PubMed2003

High-speed electrophoretic analysis of 1-phenyl-3-methyl-5-pyrazolone derivatives of monosaccharides on a quartz microchip with whole-channel UV detection.

1-Phenyl-3-methyl-5-pyrazolone (PMP) derivatives of monosaccharides were analyzed by electrophoresis on a quartz microchip with whole-channel UV detection. Rapid separation of PMP derivatives of aldopentoses was achieved by plain-zone electrophoresis in a neutral phosphate buffer with the height equivalent to a theoretical plate at the micrometer level. Zone electrophoresis as borate complexes was also successful for the separation of PMP derivatives of a few aldoses, which were separated within 1 min. Separation by microchip electrophoresis was compared to that by capillary electrophoresis, and the difference was discussed in terms of column efficiency and sample column capacity.

Antipyrine↗