Fumio Takagi

A novel high-speed droplet-polymerase chain reaction can detect human influenza virus in less than 30 min.

BACKGROUND The polymerase chain reaction (PCR) has been widely used for diagnosis of infection. Rapid detection of influenza virus is useful for therapeutic decisions. We attempted to develop a novel assay by real-time droplet-PCR machine for influenza virus. METHODS RNA extracted from nasal swabs or primary swabs pretreated only were used for PCR analyses. We evaluated reaction time, amplification efficiency, sensitivity, and specificity of the novel droplet-PCR. RESULTS The reaction time of the novel droplet-PCR was 28 min, whereas that of PCR using the conventional PCR machine was 80 min. The standard curve constructed from the amplification plots by the novel droplet-PCR was: y=-3.6x+42.9; that by PCR using the conventional PCR machine was: y=-3.5x+37.8. The sensitivity and specificity of the novel droplet-PCR were 86.7% and 91.7% for the influenza A and 100.0% and 100.0% for the influenza B, respectively. The novel droplet-PCR provided the specific amplification when using primary swabs without RNA extraction. CONCLUSIONS Our novel droplet-PCR markedly reduced the reaction time while showing same reactivity as that by PCR using the conventional PCR machine. Thus, the novel droplet-PCR assay can be used as a rapid assay for detection of influenza virus.

Rapid detection of PML–RARA fusion gene by novel high-speed droplet-reverse transcriptase-polymerase chain reaction: Possibility for molecular diagnosis without lagging behind the morphological analyses

BACKGROUND Acute promyelocytic leukemia (APL) is an aggressive disease requiring prompt diagnosis and treatment. Rapid detection of the PML-RARA fusion gene provides the molecular basis for a highly effective therapy with all-trans retinoic acid. We developed a rapid assay by novel droplet-reverse transcriptase-polymerase chain reaction (droplet-RT-PCR) for the detection of the PML-RARA fusion gene in APL patients. METHODS RNA was extracted from 7 samples obtained from 5 APL patients with the PML-RARA fusion gene confirmed by nested RT-PCR and fluorescence in situ hybridization. Using these 7 samples, we evaluated the reaction time and amplification efficiency of the droplet-RT-PCR. RESULTS Using the droplet-RT-PCR, we could detect the PML-RARA fusion gene in all 7 samples. The reaction time for 50 cycles of droplet-RT-PCR was 27 min. The amplification by the droplet-RT-PCR assay was considered positive for the PML-RARA fusion gene in less than 22 min, at the point when the fluorescence exceeded the threshold level. CONCLUSIONS Our novel droplet-RT-PCR assay is specific for the detection of the PML-RARA fusion gene and has a markedly reduced reaction time. Thus, the novel droplet-RT-PCR assay contributes to the rapid diagnosis of APL without lagging behind the morphological assessment.

Pico liter dispenser with 128 independent nozzles for high throughput biochip fabrication

This paper presents the development of the electrostatically driven inkjet-type dispenser for biochip printing. It has 128 independent reservoirs and the same number of nozzles. It is designed based on SEAJet/sup /spl reg// printhead developed for the POS printer. SEAJet/sup /spl reg// technology is suitable for dispensing bio-chemical reagent, because there is no heat generation and no use of harmful material to bio chemical molecules. In addition to the SEAJet/sup /spl reg// fabrication technology, a new process to fabricate micro through-holes and biocompatible polymer coating are developed. The dispenser achieved homogeneous and stable dispensing of protein solution. The typical droplet volume and droplet speed are 15 pico liters and 7 m/sec respectively.

Mechanical properties of hot‐rolled Pr‐Fe‐B‐Cu magnets

Mechanical strength and properties of thin plates were investigated for hot‐rolled Pr‐Fe‐B‐Cu magnets. Tensile strengths of the hot‐rolled magnets were more than 23.7 kg f/mm2. Bending tests were also applied and the strength of 37.7 kg f/mm2 was obtained. The strengths obtained are definitely higher than that of commercial Nd‐based sintered magnets. It was found that the mechanical strengths were enhanced by Pr2Fe14B grain refinement caused by Cu addition. Related to high mechanical strength, hot‐rolled Pr‐Fe‐B‐Cu magnets show excellent machinability. Thin plate magnets up to 0.1 mm in thickness with fairly large surface area were produced without any cracking. Moderate energy products better than 20 MGOe were achieved by heat treatment after machining.

Novel high-speed droplet-allele specific-polymerase chain reaction: Application in the rapid genotyping of single nucleotide polymorphisms

BACKGROUND Single nucleotide alterations such as single nucleotide polymorphisms (SNP) and single nucleotide mutations are associated with responses to drugs and predisposition to several diseases, and they contribute to the pathogenesis of malignancies. We developed a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) with our droplet-PCR machine (droplet-AS-PCR). METHODS Using 8 SNP loci, we evaluated the specificity and sensitivity of droplet-AS-PCR. Buccal cells were pretreated with proteinase K and subjected directly to the droplet-AS-PCR without DNA extraction. The genotypes determined using the droplet-AS-PCR were then compared with those obtained by direct sequencing. RESULTS Specific PCR amplifications for the 8 SNP loci were detected, and the detection limit of the droplet-AS-PCR was found to be 0.1-5.0% by dilution experiments. Droplet-AS-PCR provided specific amplification when using buccal cells, and all the genotypes determined within 9 min were consistent with those obtained by direct sequencing. CONCLUSIONS Our novel droplet-AS-PCR assay enabled high-speed amplification retaining specificity and sensitivity and provided ultra-rapid genotyping. Crude samples such as buccal cells were available for the droplet-AS-PCR assay, resulting in the reduction of the total analysis time. Droplet-AS-PCR may therefore be useful for genotyping or the detection of single nucleotide alterations.