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.

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.

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.

Spermatogenesis in aged rats after prenatal 3,3',4,4',5-pentachlorobiphenyl exposure.

We previously reported that prenatal exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126) had dose-related adverse effects on the spermatogenesis of 7(pubescent)- and 17(adult)-week-old rats, but the effects in middle and old age have been unclear. In this study, the spermatogenesis of male Sprague-Dawley rats whose dams had been injected (i.g.) with 25 pg, 2.5 ng, 250 ng, or 7.5 microg of PCB126/kg or the vehicle on days 13-19 post-conception was investigated at 52 and 90 weeks of age. At 52 weeks, the 7.5 microg group showed a significant decrease of preleptotene spermatocytes in stages VII-VIII seminiferous tubules, round spermatids increased at stages VI-VII and elongated spermatids decreased at stage VIII, while the spermatogenesis of the other PCB-treated groups were similar to that of the vehicle group. At 90 weeks, the 7.5 microg group showed a significant decrease of spermatogenic cells at many stages, and the 250 ng group showed a significant decrease of preleptotene spermatocytes at stages VII-VIII, and round spermatids increased at stages VI-VII, elongated spermatids decreased at stage VIII, and the spermatogenesis of the 2.5 ng and 25 pg groups were similar to those of the vehicle group. The present study showed that prenatal PCB126 exposure had dose-related adverse effects on spermatogenesis in aging rats and may have accelerated spermatogenic senescence. Because the serum testosterone levels of the PCB126 groups and the vehicle group were similar, a direct endocrine cause for the observed effects was unlikely.

Biocompatible inkjet printing technique for designed seeding of individual living cells.

Inkjet printers are capable of printing at high resolution by ejecting extremely small ink drops. Established printing technology will be able to seed living cells, at micrometer resolution, in arrangements similar to biological tissues. We describe the use of a biocompatible inkjet head and our investigation of the feasibility of microseeding with living cells. Living cells are easily damaged by heat; therefore, we used an electrostatically driven inkjet system that was able to eject ink without generating significant heat. Bovine vascular endothelial cells were prepared and suspended in culture medium, and the cell suspension was used as "ink" and ejected onto culture disks. Microscopic observation showed that the endothelial cells were situated in the ejected dots in the medium, and that the number of cells in each dot was dependent on the concentration of the cell suspension and ejection frequency chosen. After the ejected cells were incubated for a few hours, they adhered to the culture disks. Using our non-heat-generating, electrostatically driven inkjet system, living cells were safely ejected onto culture disks. This microseeding technique with living cells has the potential to advance the field of tissue engineering.