• Journal of Life Science
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• v.20 no.12
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• pp.1896-1901
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• 2010

This study was conducted using quantitative real-time PCR using Lactobacilli as probiotics. Quantitative real-time PCR (RT PCR) was conducted via a method involving SYBR Green 1 and a probe. Plasmid DNA was cloned using the 16S-23S rRNA intergenic species region. Gene clones were diluted from $10^2$ to $10^{10}$. Standard curves were constructed via Ct values obtained from the results of Real-time PCR via the aforementioned SYBR Green 1 and probe method. Plasmid DNA was also cloned using the 16S-23S rRNA intergenic species region and the gene clones were diluted from $10^2$ to $10^{10}$ copy numbers via the probe method. Using RT PCR, a standard curve of plasmid DNA copy numbers was also determined. The slope value for the Y-axis intercept and $R^2$ value were measured as -3.346, 33.18, and 0.993, respectively, via the first method. For the second method, the slope value for the Y-axis intercept and $R^2$ were -3.321, 31.10 and 0.995, respectively. The PCR inhibitor could not express the detection curve at a copy number over $10^{10}$ via either method, owing to high DNA density. The DNA extract from probiotics was diluted without pre-culturing, and 16 products were amplified via both methods. The Ct value was 11.06~18.12 in the first method and 16.74~22.11 in the second method. Measured probiotics and log copy values were largely similar among the methods used. It was concluded that both methods are effective for analysis, but further research will be required to verify the optimal method.

• Food Science of Animal Resources
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• v.37 no.6
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• pp.799-803
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• 2017

In the present study, centrifugation and filtration pretreatments were evaluated to decrease sample preparation time and to improve the sensitivity and specificity of multiplex polymerase chain reaction (PCR) for the detection of low levels of pathogenic Escherichia coli in various foods. Pathogenic E. coli (E. coli NCCP11142, E. coli NCCP14037, E. coli NCCP 14038, E. coli NCCP14039, and E. coli NCCP15661) was inoculated into pork, beef, and baby leafy vegetables at 1, 2, and 3 Log CFU/g. The samples were shaken 30 times (control), then centrifuged or filtered. DNA extracts from the samples were subjected to PCR using the $Powerchek^{TM}$ Diarrheal E. coli 8-plex Detection Kit. In the pork samples, no E. coli was detected in the control samples, while E. coli were detected in 100% of 3-Log CFU/g inoculated and centrifuged samples, and in 100% of 2 and 3-Log CFU/g inoculated, and filtered samples. In the beef samples, all control samples appeared to be E. coli-negative, while E. coli was detected in 50-75% of centrifuged samples, regardless of inoculated level, and in 100% of 2 and 3-Log CFU/g inoculated, and filtered samples. In baby leafy vegetables, E. coli were not detected in 25-50% of the control samples, while E. coli were detected in 0-25% of the centrifuged samples, and 75-100% of the filtered samples, depending on the inoculum amount. In conclusion, filtration pretreatment can be used to minimize sample preparation time, and improve the sensitivity and specificity of rapid detection of pathogenic E. coli in various foods.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.177-181
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• 2003

Eleven bacterial strains which are able to utilize terephthalic acid as a carbon and an energy source for growth were isolated from the soil of 7 water quality evaluation points in Kyonggi area of Korea. Phthalic acid isomer degrading activity of the isolates from the 4 contaminated points was higher than those from the 3 clean points. Among 11 isolates, 4 isolates which have high terephthalic acid degrading activity and degrade two phthalic acid isomers were identified by partal 16S rDNA sequence determination. One of them was identified as Pseudomonas putida, and the others as Comamonas testosteroni. Thus a large number of phthalic acid isomer degrading bacteria in domestic soil were inferred as C. testosteroni. On the basis of these results, the PCR detection of C. testosteroni in soil was applied to monitor soil contamination by phthalic acid isomers. The DNA of C. test-osteroni extracted from 4 g soil was directly detected by PCR with C. testosteroni specific primer pair. The amount of PCR products was different according to sampling sites and more PCR products were obtained from contaminated sites than those from clean sites (Gulpo-chun＞Anyang-chun＞Hwangguji-chun>Shin-chun＞Huk-chun＞Pukhan-river>Kapyeong-chun). This result was coincided with that of the viable cell counts for terephthalic acid degrading bacteria.

• Journal of the East Asian Society of Dietary Life
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• v.24 no.6
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• pp.739-747
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• 2014

The aim of the study was to determine the effects of Ixeris dentata extract (IDE) on anticancer activity in human breast cancer MDA-MB-231 cells at both cellular and molecular levels. The cells were cultured in the presence of 0, 20, 30 and $40{\mu}g/mL$ Ixeris dentata extract for 24 hours, respectively. At the end of culture, cytochemical analyses for MTT activity, trypan blue dye exclusion, Annexin V-FITC Apoptosis, and radical oxygen species (ROS) were conducted. RT-PCR was also performed to determine whether or not alterations in cell viability affect the Bax/Bcl-2 ratio. MTT assay showed that relative cell viability decreased in a dose-dependent manner (p<0.05). Reduction of cell viability matched well with increased cell membrane permeability as determined by trypan blue dye exclusion test (p<0.05). The rates of intracellular ROS also increased in a similar manner to those of TB-stained cells. There was an associated shift of apoptotic cells from early to late apoptosis between the 30 and $40{\mu}g/mL$. Bax/Bcl-2 ratio significantly increased along with significant decreases in Bcl-2 expression between 30 and $40{\mu}g/mL$ groups (p<0.05). In conclusion, anticancer activity of Ixeris dentata extract is modulated by a reduction in cell viability along with increased membrane permeability, leading to ROS accumulation within cells, and subsequently cell death through an apoptotic pathway that involves Bax and Bcl-2 in human breast cancer MDA-MB-231 cells.

• The Plant Pathology Journal
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• v.30 no.3
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• pp.304-309
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• 2014

The rpf genes and $colS_{XOO1207}/colR_{XOO1208}$ were known to require for virulence of Xanthomonas oryzae pv. oryzae (Xoo). In Xoo KACC10331 genome, two more colS/colR genes, $colS_{XOO3534}$ (raxH)/$colR_{XOO3535}$ (raxR) and $colS_{XOO3762}/colR_{XOO3763}$ were annotated. The $colS_{XOO3534}/colR_{XOO3535}$ were known to control AvrXa21 activity and functions of $colS_{XOO3762}/colR_{XOO3763}$ were unknown in Xoo. To characterize the relationship between rpf and colS/colR genes, expression of colS/colR genes in Rpf mutants of Xoo were analyzed with quantitative reverse transcription PCR (qRT-PCR). Expressions of all three colS/colR genes increased in the rpfF mutant in which DSF synthesis is defective. Expression of $colS_{XOO1207}/col-R_{XOO1208}$, $colS_{XOO3534}/colR_{XOO3535}$ and $colS_{XOO3762}/colR_{XOO3763}$ increased 2, 2-7, 3-13 folds respectively. Expression of $colS_{XOO3534}$ and $colS_{XOO3762}$ also increased 2-4 folds in the rpfG mutant in which the signal from DSF is no longer transferred to down-stream. Expression of the other colS/colR genes was not significantly changed in the rpfG mutant compared to the wild type. Since RpfF and RpfG are responsible for DSF synthesis and signal transfer from DSF to down-stream to regulate virulence gene expression, these results suggest that the DSF and DSF-mediated signal regulate negatively three colS/colR genes in Xoo.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.171-180
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• 2001

Compared to other gene transfer system, the advantages of retrovirus-mediated gene transfer are technical ease, efficient expression and genetic stability. Despite the high potency of the retrovirus vector system in gene transfer, one of the drawbacks is a difficulty in concentration of virus stock. To overcome this problem, we tested a new retrovirus vector system producing the progeny retrovirus particles encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein). The infectivity of this virus was not sacrificed by ultracentrifugal concentration and the host cell range extended from all mammalian to fish embryos. Virus titer after 1,000 x concentration was more than 10$^{8}$ CFU/ $m\ell$ on most of the target cell lines. We applied this pantropic viruses in transgenic chicken production by injecting the concentrated (100$\times$) stock into subgerminal cavity of stage X chicken embryos. The survival rate of chicken embryos after injection was about 20% and gene integration rate in surviving embryos was scored almost 100%. Analyses of RT-PCR and fluorescence microscopy, however, showed no evidence of the transgene expression.

• Journal of Animal Science and Technology
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• v.49 no.5
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• pp.569-576
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• 2007

receptor(MC4R) gene and carcass traits was examined in randomly selected commercial pigs. A porcine MC4R gene was genotyped for Asp298Asn(nt. 892G>A) by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). A total of three genotypes, A/A, A/G, and G/G, were found with 28.8, 22.8, and 48.4% frequencies, respectively. In the whole population, pigs containing 892A/- showed significantly higher marbling score than those of homozygotes G/G(P<0.05). Two homozygotes, A/A and G/G showed lower in meat color score but higher in water holding capacity than those of heterozygotes A/G(P<0.01). However, the carcass weight of the barrows containing wild type -/G was significantly higher(i.e. more than 2.5kg) than those of homozygotes A/A(P<0.05). The effects of each genotype on carcass traits in the gilts were similar to those of the whole population, but not in barrows, suggesting an unknown sex-related effect on carcass traits. This study suggested that the genotype MC4R A/- could improve the meat quality in the commercial pig production. However, since the genetic polymorphism of MC4R gene differentially affected the carcass traits in sex-related manner, therefore, both parameters, the sex and genotype, should be considered for marker-assisted selection in commercial pig production.

• Journal of Microbiology and Biotechnology
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• v.33 no.8
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• pp.998-1005
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• 2023

Streptococcus salivarius is a beneficial bacterium in oral cavity, and some strains of this bacterium are known to be probiotics. The purpose of this study was to investigate the anti-inflammatory effect and mechanism of S. salivarius G7 lipoteichoic acid (LTA) on lipopolysaccharide (LPS) and LTA of periodontopathogens. The surface molecules of S. salivarius G7 was extracted, and single- or co-treated on human monocytic cells with LPS and LTA of periodontopathogens. The induction of cytokine expression was evaluated by real-time PCR and ELISA. After labeling fluorescence on LPS and LTA of periodontopathogens, it was co-treated with S. salivarius LTA to the cell. The bound LPS and LTA were measured by a flow cytometer. Also, the biding assay of the LPS and LTA to CD14 and LPS binding protein (LBP) was performed. The surface molecules of S. salivarius G7 did not induce the expression of inflammatory cytokines, and S. salivarius G7 LTA inhibited the inflammatory cytokines induced by LPS and LTA of periodontopathogens. S. salivarius G7 LTA inhibited the binding of its LPS and LTA to cells. Also, S. salivarius G7 LTA blocked the binding of its LPS and LTA to CD14 and LBP. S. salivarius G7 has an inhibitory effect on inflammation induced by LPS or LTA of periodontopathogens, and may be a candidate probiotics for prevention of periodontitis.

• Parasites, Hosts and Diseases
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• v.34 no.2
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• pp.127-134
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• 1996

Twelve isolates of Accnthamoebc app. assigned to either A. castellanii or A. poIMphoSa, and type strains of A. culbefsoni, A. henIWi, A. pqkestinefiE, and A. astronyxi,s were examined by restriction fragment length polymorphism (RFLP) of a conserved region of small subunit ribosomal RMh gene (ssu rDNA) amplified by polymerase chain reaction (PCR). The PCR products of the isolates measured approximately 910-930 bp, except for that of A. astronyxis which was extraordinarily long, approximately 1,170 bp. Average of estimated sequence divergence of the amplified DNA among the isolates assigned to A. castellanii was 9.8% whereas that among the isolates assigned to A. polvphusn 9.6%. The maximum intraspecific sequence divergence among the isolates assigned to A. costellanii was observed between the Chang and Ma strains (17.3%) while that among the isolates assigned to A. poIWphosa was observed between KA/S3 and KA/S7 strains (16.1%). The both maximum sequence divergences were much greater than the minimum interspecific sequence divergence between A. cnstellnnii and A. polwphasa (2.6%) which appeared between the Castellani (or CCAP 1501/12 g) and KA/S3 strains. The PCR-RFLP patterns of A. culbertsoni, A. healyi, A. palestinensis, and A. ostronvxis were quite diverse from one another and from those of isolates assigned to either A. castellanii or A. polyphoga. It is suggested that taxonomic validity of the isolates assigned to either A. castellnnii or A. polyphoga should be reevaluated.

• Mycobiology
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• v.47 no.3
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• pp.350-354
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• 2019

In the post-genomic era, gene function analysis has attracted much attention. Transformation is often needed to investigate gene function. In this study, an easy, rapid, reliable, and cost-effective colony polymerase chain reaction (PCR) method for screening mushroom transformants was developed: picking up a suitable amount of transformant's tissue ($1-10{\mu}g$) to $20{\mu}l$ 0.25% Lywallzyme solution, and vortexing for 10 s followed by incubation at $34^{\circ}C$ for 15 min. Finally, $2{\mu}l$ of the suspension was used as templates to perform PCR and single target bands were successfully amplified from respective transformants of Tremella fuciformis, Pleurotus ostreatus, and Pleurotus tuber-regium. This procedure could be widely employed for screening transformants in mushroom transformation experiments.