• Biomedical Science Letters
• /
• v.22 no.3
• /
• pp.83-97
• /
• 2016

The measurement of viral load in HIV-1 infected patients is essential for the establishment of a therapeutic strategy. Several commercial assays have shown shortcomings in quantifying rare genotypes of HIV-1 such as minor groups of N and O. In this study, the HIV-1 RT-qPCR assay was developed. The primers and probe of HIV-1 were designed to target the pol gene and to increase the detection efficiency of various subtypes including group N and O. The HIV-1 quantitative RT-qPCR assay was assessed for its analytical performance and clinical evaluation. The LoD was determined to 33.9 IU/ml. The LoD of several subtypes including A, C, D, CRF_01AE, F, CRF_02AG, G and H, were determined to less than 40 IU/ml. The HIV-1 quantitative RT-qPCR assay was evaluated using the China National Reference Panel of HIV-1 RNA to determine the analytical performance. The results were all within the acceptable range. The clinical evaluation was performed at Hunan CDC in China. The clinical evaluation results were compared with those of the China domestic commercial kit. A significant correlation (fresh samples; $R^2=0.84$, P<0.001, frozen samples; $R^2=0.76$, P<0.001) between the two systems was observed for 64 fresh samples and 76 frozen samples with viral loads, and the Bland-Altman plot showed good agreement (98.4%, 96.1%, respectively). In conclusion, the HIV-1 quantitative RT-qPCR assay had comparable analytical performance with several commercial kits. The study provides basic data for the research of HIV-1 diagnosis and the development of P < HIV-1 molecular diagnostic assay.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.46 no.9
• /
• pp.1097-1105
• /
• 2017

This study examined how much chicken meat was in sausage made with pork. Both real-time polymerase chain reaction (PCR) and internal standard addition were used. Fifty ng of chicken DNA was added to the sausages as an internal standard. The addition of standard DNA increased the amplification efficiency of PCR and confirmed the possibility of quantitative analysis. A QIAamp DNA Micro Kit was used to improve the DNA recovery and amplification efficiency. The density of template DNA and primer were suitable for $3.0{\sim}5.0{\mu}L$ and $0.5{\mu}L$, respectively. Each DNA of pig and chicken was diluted in 10-fold from steps 50 ng to 0.05 ng. The detection limit of both pig and chicken meat was more than 0.05 ng and the correlation coefficient of the standard curve was at least 0.98. The result of the quantitative analysis after heat treatment of 3 samples of pigs and chickens mixed at 70:30 showed a 5.7% difference (64.3:35.7) between the expected value and measured value. The quantitative value was changed by affecting the DNA according to the heat treatment ($70^{\circ}C$, 10 min). An analysis of the pork and chicken content in sausages showed that it was difficult to detect chicken meat and the quantitative value of DNA according to the Ct value was very low. On the other hand, when adding standard material (50 ng of chicken DNA) to the sausages, the Ct value decreased gradually with increasing chicken mixing ratio. Thus, the mixing ratio of chicken in sausages could be estimated.

• Korean Journal of Food Science and Technology
• /
• v.37 no.6
• /
• pp.1018-1023
• /
• 2005

Simplified procedure was developed for concentrating and detecting poliovirus and norovirus in oysters. Viruses were seeded into oyster tissue homogenates and concentrated through polyethylene glycol (PEG) precipitation, chloroform or Freon extraction, with additional PEG precipitation. Amount of viruses was evaluated using poliovirus plaque assay. Virus recovery during concentration procedure was approximately 16.4-26.0%. For defection, viral RNAs in oysters were examined using one-step RT-PCR after extraction with Trizol. Dilution or capturing of viral RNA using silica gel membrane allowed viruses to be detected by RT-PCR, whereas viruses could not be removed using $QIAshredder^{TM}$ Homogenizer, which is effective in removing RT-PCR inhibitors in lettuce and hamburgers. Freon extraction, generally used to concentrate viruses found in food, could be substituted with chloroform extraction using this procedure; no difference could be observed between detection limits of whole oyster extracts and digestive organ extracts indicating that RT-PCR inhibitors were distributed evenly throughout whole tissues. Nested PCR greatly improved efficiency of this procedure. Overall, this procedure could remove sufficient amount of inhibitors to allow detection of norovirus in oysters.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2006.10a
• /
• pp.56-56
• /
• 2006

• Korean Journal of Plant Tissue Culture
• /
• v.27 no.2
• /
• pp.95-100
• /
• 2000

The cytosolic glutathione reductase (GR) gene of Brassica campestris L. was introduced into several Japonica cultivars of rice by Agrobacterium tumefaciens and a large number of transgenic plants were produced. Three-week old calli were co-cultivated with A. tumefaciens strain EHA101 carrying the plasmid pIGR1. The efficiency of transformation was differed from rice cultivars. A Japonica cultivar, 'Daeribbyeo' appeared the highest efficiency (42.5%) of transformation among the four cultivars tested. The addition of acetosyringone (50 $\mu$M) during co-cultivation was a key to successful transformation. Transgene fragments were identified by PCR amplification and further confirmed by Southern blot analysis. Mendelian inheritance of the transgenes was confirmed in T$_1$ progeny.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.4
• /
• pp.479-483
• /
• 2012

The secretory efficiency of recombinant xylanase xynB from yeast Pichia pastoris between the ${\alpha}$-factor preprosequence and a classical mammalian signal peptide derived from bovine ${\beta}$-casein was compared. The results showed that although the bovine ${\beta}$-casein signal peptide could direct high-level secretion of recombinant xylanase, it was relatively less efficient than the ${\alpha}$-factor preprosequence. In contrast, the bovine ${\beta}$-casein signal peptide caused remarkably more recombinant xylanase trapped intracellularly. Real-time RT-PCR analysis indicated that the difference in the secretory level between the two signal sequences was not due to the difference in the transcriptional efficiency.

• Journal of Life Science
• /
• v.20 no.8
• /
• pp.1173-1180
• /
• 2010

We investigated changes in photosynthesis and activity of ascorbate peroxidase (APX) that scavenges ROS as responses to oxidative stress induced by salinity in rice (Oryza sativa L.). Photosynthetic efficiency of rice leaves, monitored in terms of Fv/Fm, declined with the increase of salt concentration (100-300 mM NaCl). Salinity caused an increase of $H_2O_2$ in leaves of rice, with an increase of APX activity. Among total APX isoforms, an isoform of stromal-APX 1 in leaves of rice was completely inactivated by 300 mM NaCl, but was not affected by chilling or drought. The results suggest that salt stress acts in quite a different mechanism in relation to the activity of stromal-APX from that of other stresses such chilling and drought. We carried out RT-PCR for analysis of genes expression of APX isoforms as affected by salt stress. The expression of cytosolic APX/thylakoid-bound APX genes in leaves of rice exposed to salt stress was increased, while stromal APX gene expression rapidly declined.

• International Journal of Oral Biology
• /
• v.37 no.4
• /
• pp.181-188
• /
• 2012

As the demand for large-scale analysis of gene expression using DNA arrays increases, the importance of the surface characterization of DNA arrays has emerged. We compared the efficiency of molecular biological applications on solid-phases with different surface polarities to identify the most optimal conditions. We employed thiol-gold reactions for DNA immobilization on solid surfaces. The surface polarity was controlled by creating a self-assembled monolayer (SAM) of mercaptohexanol or hepthanethiol, which create hydrophilic or hydrophobic surface properties, respectively. A hydrophilic environment was found to be much more favorable to solid-phase molecular biological manipulations. A SAM of mercaptoethanol had the highest affinity to DNA molecules in our experimetns and it showed greater efficiency in terms of DNA hybridization and polymerization. The optimal DNA concentration for immobilization was found to be 0.5 ${\mu}M$. The optimal reaction time for both thiolated DNA and matrix molecules was 10 min and for the polymerase reaction time was 150 min. Under these optimized conditions, molecular biology techniques including DNA hybridization, ligation, polymerization, PCR and multiplex PCR were shown to be feasible in solid-state conditions. We demonstrated from our present analysis the importance of surface polarity in solid-phase molecular biological applications. A hydrophilic SAM generated a far more favorable environment than hydrophobic SAM for solid-state molecular techniques. Our findings suggest that the conditions and methods identified here could be used for DNA-DNA hybridization applications such as DNA chips and for the further development of solid-phase genetic engineering applications that involve DNA-enzyme interactions.

• Mycobiology
• /
• v.47 no.1
• /
• pp.59-65
• /
• 2019

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (${\leq}0.1%$) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per $10^6$ YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.

• Journal of Korean Society of Forest Science
• /
• v.105 no.4
• /
• pp.422-428
• /
• 2016

Ginkgo (Ginkgo biloba L.) is one of the most appropriate roadside trees because of a good transplantation nature and ability to grow well in urban environment. Ginkgo is a dioecious species. Sex discrimination of ginkgo is possible through comparing morphological characters of reproductive organs. However, it needs more than about twenty years for reproductive organs to appear after sexual maturity. Until now, ginkgo trees for roadside plantation have been planted without discriminating the sex because ginkgo trees have been usually planted before sexual maturity. Ginkgo nuts from the female ginkgo trees planted along the roadside emit a foul odor, and make much pollution on the streets. Thus in this study a novel SCAR marker (SCAR-GBM) for the early sex discrimination was developed. Primers were developed on the basis of the sequence of male-specific RAPD variants reported previously. False-negative problem of SCAR marker, probably caused by dominant nature, was resolved by using multiplex PCR using primers of both the SCAR-GBM and a universal primer set of atp1 region in mitochondria DNA, which resulted in improved discrimination efficiency. The results showed that DNA bands of 1,039 bp were commonly amplified by the atp1 primer set in male and female trees, and SCAR-GBM markers of 675 bp were specifically amplified only in male trees. Reproducible and specific discrimination of the multiplex PCR was finally confirmed by applying multiple male and female individuals.