Akemi Yamaguchi

Effects of Semiconductor Thickness on Poly-Crystalline Silicon Thin Film Transistors.

The effects of semiconductor thickness on poly-crystalline silicon (poly-Si) thin film transistors (TFTs) are studied. The small value of the threshold voltage (V th ) appearing in thin film can improve the electrical properties of the corresponding TFTs. However, thin poly-Si film generally consists of small grains and contains many defects. When the film is extremely thin, it contains voids and it is in a separate island-like state. As a result, the extremely thin film possesses very many traps, which prevent the V th value from further decreasing. The way that thickness affects film quality differs according to how that film was prepared. Therefore, different TFT fabrication methods make the optimum semiconductor thickness different.

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.

Evaluation of saliva as diagnostic materials for influenza virus infection by PCR-based assays.

BACKGROUND Immunochromatographic antigen tests have been widely used for detection of influenza virus; however its low sensitivity restricts the use of clinical materials other than nasopharyngeal swabs. Saliva is obtained non-invasively and has utility for diagnosis of influenza. Polymerase chain reaction (PCR) is not typically used for rapid testing because it is time consuming. We evaluated the utility of saliva as diagnostic materials for influenza virus infection by PCR-based assays. METHODS Nasopharyngeal swabs and saliva were simultaneously collected from 144 patients and investigated by reverse transcription-quantitative PCR (RT-qPCR) and droplet-RT-PCR. RESULTS Overall concordance of results from nasopharyngeal swabs and saliva were 95.8%. Influenza gene was detectable in less than 12min in saliva by the droplet-RT-PCR. Saliva as well as nasopharyngeal swabs contained more than 1×10(2) copies/μl of the influenza gene. About half of the patients provided positive results in nasopharyngeal swabs and saliva within 24h from the onset of the symptoms. CONCLUSION The study demonstrates that saliva can be used as an alternative specimen source to nasopharyngeal swabs. When rapid PCR assay including RNA extraction to be full-automation in a miniaturized machine, point-of-care test based on PCR may be realized using saliva without restriction of materials.

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR

BACKGROUND Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. METHODS SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). RESULTS Droplet-AS-PCR could determine genotypes within 8min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. CONCLUSION The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.

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.

A Rapid and Automated Device for Purifying Nucleic Acids.

We have developed a rapid, automated nucleic acid purification device in a single cartridge containing silica-coated magnetic beads. We succeeded in extracting the matrix protein gene of influenza A virus from pharyngeal swab samples within 3 min. The device will be widely applicable to detect a specific gene from the various samples for clinical diagnosis and genetic research.

Development of a rapid and sensitive one-step reverse transcription-nested polymerase chain reaction in a single tube using the droplet-polymerase chain reaction machine.

BACKGROUND Reverse transcription (RT)-nested polymerase chain reaction (PCR) is a time-consuming procedure because it has several handling steps and is associated with the risk of cross-contamination during each step. Therefore, a rapid and sensitive one-step RT-nested PCR was developed that could be performed in a single tube using a droplet-PCR machine. METHODS The K562 BCR-ABL mRNA-positive cell line as well as bone marrow aspirates from 5 patients with chronic myelogenous leukemia (CML) and 5 controls without CML were used. We evaluated one-step RT-nested PCR using the droplet-PCR machine. RESULTS One-step RT-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR-ABL mRNA within 40min, which was 10(3)-fold superior to conventional RT nested PCR using three steps in separate tubes. The sensitivity of the one-step RT-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay. CONCLUSIONS One-step RT-nested PCR was developed using the droplet-PCR machine, which enabled all reactions to be performed in a single tube accurately and rapidly and with high sensitivity. This one-step RT-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories.

Rapid diagnosis of acute promyelocytic leukemia with the PML-RARA fusion gene using a combination of droplet-reverse transcription-polymerase chain reaction and instant-quality fluorescence in situ hybridization

BACKGROUND Acute promyelocytic leukemia (APL) with the PML-RARA fusion gene can be effectively cured using molecular-targeted therapies, which require both detection and quantification of the PML-RARA fusion gene. Here, we developed a rapid assay for identifying and measuring the PML-RARA fusion gene in patients with APL using droplet-reverse transcription-polymerase chain reaction (droplet-RT-PCR) and instant quality-fluorescence in situ hybridization (IQ-FISH). METHODS RNA for droplet-RT-PCR and fixed-cell suspensions for IQ-FISH were prepared from five patients with APL and three controls. We evaluated the amplification efficiency and reaction time with droplet-RT-PCR and signal clarity and hybridization time with IQ-FISH. RESULTS The reaction using droplet-RT-PCR was completed in 26min. The PML-RARA fusion gene was detected in all samples from the five patients. IQ-FISH yielded clear signals after 1h of hybridization. There were no significant differences in signal clarity or positive signal ratios between IQ-FISH and conventional FISH. CONCLUSIONS Simultaneous droplet-RT-PCR and IQ-FISH, in addition to morphological examination of blood smears, can be used to diagnose patients as having APL within 4h based on molecular/cytogenetic results. Rapid diagnosis can allow effective therapies to be started promptly.