• Journal of Periodontal and Implant Science
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• v.48 no.4
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• pp.261-271
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• 2018

Purpose: Few studies have examined periodontal pathogens from saliva samples in periodontally healthy young adults. The purposes of this study were to determine the prevalence of periodontopathic bacteria and to quantify periodontal pathogens in saliva samples using real-time polymerase chain reaction (PCR) assays in periodontally healthy Korean young adults under 35 years of age. Methods: Nine major periodontal pathogens were analyzed by real-time PCR in saliva from 94 periodontally healthy young adults. Quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum, Campylobacter rectus, Peptostreptococcus anaerobius, and Eikenella corrodens was performed by DNA copy number measurement. Results: F. nucleatum and E. corrodens were detected in all subjects; the numbers of positive samples were 87 (92.6%), 91 (96.8%), and 90 (95.7%) for P. gingivalis, P. anaerobius, and C. rectus, respectively. Other pathogens were also detected in periodontally healthy subjects. Analysis of DNA copy numbers revealed that the most abundant periodontal pathogen was F. nucleatum, which was significantly more prevalent than all other bacteria (P<0.001), followed by P. anaerobius, P. gingivalis, E. corrodens, C. rectus, and T. denticola. There was no significant difference in the prevalence of each bacterium between men and women. The DNA copy number of total bacteria was significantly higher in men than in women. Conclusions: Major periodontal pathogens were prevalent in the saliva of periodontally healthy Korean young adults. Therefore, we suggest that the development of periodontal disease should not be overlooked in periodontally healthy young people, as it can arise due to periodontal pathogen imbalance and host susceptibility.

• Journal of Microbiology
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• v.39 no.2
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• pp.95-101
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• 2001

In an effort to develop an efficient expression and secretion system for heterologous proteins in Aspergilius nidulans, the PCR-amplified coding sequence for alkaline pretense (AlpA) of A. oryzae was cloned into a fungal expression vector downstream of A. nidulans aicA (alcohol dehydrogenase) promoter to yield pRAAlp. Transformation of A. nidulans with pRAAlp gave stable transformants harboring various copy numbers (3 to 10) of integrated alpA gene, from among which 6 representatives were selected. On a medium containing 0.8% ammonium sulfate that represses the expression of the host's own pretense, the alcA prumoter-controlled AlpA expression was strongly induced by threonine but repressed by glucose. The level of AlpA secretion was highest (approximately 666 mU/ml) in transformant ALP6 containing the largest copy number integrated alpA. However, the level of AlpA secretion was not necessarily proportional to the copy numbers of the integrated alpA genes. The N-terminal sequence or the secreted mature AlpA was determined to be Gly-Leu-Thr-Thr-Gln-Lys-Ser and its molecular mass to be approximately 34 kDa, indicating that AlpA is properly processed by the removal of 121 N-terminal amino acids.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1353-1358
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• 2005

A rapid and quantitative real-time PCR was developed to target the invasion A (invA) gene of Salmonella spp. We developed quantitative standard curves based on plasmids containing the invA gene. Based on these curves, we detected Salmonella spp. in artificially contaminated buffered peptone water (BPW) and milk samples. We were able to determine the invA gene copy number per ml of food samples, with the minimum detection limit of $4.1{\times}10^{3}$ copies/ml of BPW and $3.3{\times}10^{3}$ copies/ml of milk. When applied directly to detect and quantify Salmonella spp. in BPW and milk, the present real-time PCR assay was as sensitive as the plate count method; however, copy numbers were one to two logs higher than the colony-forming units obtained by the plate count methods. In the present work, the real-time PCR assay was shown to significantly reduce the total time necessary for the detection of Salmonella spp. in foods and to provide an important model for other foodborne pathogens.

• ALGAE
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• v.32 no.3
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• pp.189-197
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• 2017

To quantify the abundance of the harmful dinoflagellate Cochlodinium polykrikoides in natural seawaters, we developed the innovative procedure using a droplet digital PCR (ddPCR) with C. polykrikoides-specific primers targeting the internal transcription sequence (ITS). The abundance of C. polykrikoides was estimated by the specific copy number of target ITS DNA segments per cell in cultures and natural water samples. The copy number per C. polykrikoides cell as acquired by ddPCR was $157{\pm}16$, which was evaluated against known cell numbers through a simplified protocol preparing DNAs. The abundances of C. polykrikoides in the waters of different locations estimated by ddPCR agreed with the number of cells visually counted under a microscope. This protocol was used to measure the abundance of C. polykrikoides close to and further off the southern coast of Korea in August of 2016 and 2017. The practical application showed that this method can reduce time for analysis and increase accuracy.

• Journal of fish pathology
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• v.34 no.2
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• pp.161-168
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• 2021

Quantitative analysis of myxosporean parasites (Enteromyxum leei and Parvicapsula anisocaudata) were performed using real-time PCR on the internal organs (head kidney, body kidney, intestine, spleen, brain, liver, heart, muscle, blood, and eye) of emaciated Paralichthys olivaceus from farm-A. The highest DNA copy number of E. leei was shown in the intestine (1.3 × 10⁸ copies/mg tissue) of emaciatied P. olivaceus and DNA copy number in the other internal organs (1.3 × 10³~4.6 × 10⁵ copies/mg tissue) showed lower than in intestine. From the result of real-time PCR for P. anisocaudata, it was considered mildly infected, due to the low DNA copy numbers of the head kidney (1.3 × 10³ copies/mg tissue) and body kidney (9.1 × 10³ copies/mg tissue). In order to investigate whether myxosporean parasites can be detected in a non-invasive way, quantitative analysis of E. leei and P. anisocaudata from rearing water of three farms were performed by real-time PCR. The DNA copy number of E. leei from rearing water of farm-A and farm-B were 8 × 10⁴ and 5 × 10⁵ copies/L, respectively. However, it was not detected in farm-C. For P. anisocaudata from rearing water, farm-A, farm-B and farm-C showed 0, 2.0 × 10⁶ and 5.1 × 10⁶ copies/L, respectively.

• Journal of Korean Academy of Dental Administration
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• v.10 no.1
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• pp.42-52
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• 2022

This study aimed to investigate the association of lifestyle with the copy number of periodontal pathogens. This retrospective study collected electronic health records of 102 subjects with periodontitis, including reports of bacterial genetic tests and lifestyle questionnaires. The five pathogens were analyzed as follows: Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, and Fusobacterium nucleatum. The lifestyle questionnaire included age, sex, oral hygiene management, smoking, drinking, exercise, dietary, snacks, water intake, and sleeping time. An independent t-test or ANOVA was performed to compare the copy number of periodontal pathogens according to lifestyle (α=0.05). The copy numbers of P. gingivalis and F. nucleatum were significantly higher than those of other strains. The copy number of T. forsythia in patients who exercised was 54% lower than in those who did not (p=0.009). Other lifestyle factors did not affect the number of bacteria. Exercise habits among the lifestyles showed a association with the number of specific oral bacteria. This result suggests that a lifestyle questionnaire is essential in clinical situation and necessary to prevent and treat the periodontal disease effectively.

• Journal of fish pathology
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• v.36 no.2
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• pp.229-238
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• 2023

In this study, the correlation between clinical changes and RSIV genome copy number was investigated to determine the quantitative criteria for the characteristics of RSIV infection. The rock bream (Oplegnathus fasciatus) was intraperitoneally injected with three different doses (1.0×10¹, 1.0×10³ and 1.0×10⁵ viral genome copies/fish) as low, medium, and high doses, respectively. The clinical signs (spleen enlargement, death) observation and real-time PCR were conducted at 5, 10 and 14 days post-injection. During the experiment, spleen index as a quantitative indicator for spleen enlargement was continuously increased in the medium- (up to 2.26) and high-dose (up to 4.99) challenge groups, respectively. Notably, when the spleen index was over 1.5, 2.0, 2.5 and 3.0, a positive correlation was revealed with average viral genome copy numbers of 2.51, 3.37, 4.97 and 5.43×10⁷ viral genome copies/mg, respectively. Moreover, the threshold of spleen index over 1.5 was 1.0×10⁶ viral genome copies/mg, while the thresholds of spleen index over 2.0 and dead was 2.51×10⁷ viral genome copies/mg and the thresholds of spleen index over 2.5 and 3 was 3.98×10⁷ viral genome copies/mg. These findings suggest the possibility of quantitatively analyzing the characteristics and development process of RSIV infection.

• Journal of Soil and Groundwater Environment
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• v.21 no.4
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• pp.50-59
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• 2016

Spatial and temporal changes in nitrification activities and distribution of microbial population of ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) in paddy soils were investigated. Soil samples were collected in March and October 2015 from rice paddy with and without the presence of confined animal feeding operations. Incubation experiments and quantitative polymerase chain reaction showed that AOA's contribution to nitrification kinetics was much higher in locations where organic nitrogen in animal waste is expected to significantly contribute to overall nitrogen budget, and temporal variations in nitrification kinetics were much smaller for AOA than AOB. These differences were interpreted to indicate that different microbial responses of two microbial populations to the types and concentrations of nitrogen substrates were the main determining factors of nitrification processes in the paddy soils. The copy numbers of ammonium monooxygenase gene showed that AOA colonized the paddy soils in higher numbers than AOB with stable distribution while AOB showed variation especially in March. Although small in numbers, AOB population turned out to exert more influence on nitrification potential than AOA, which was attributed to higher fluctuation in AOB cell numbers and nitrification reaction rate per cells.

• Microbiology and Biotechnology Letters
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• v.21 no.3
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• pp.214-220
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• 1993

The mating type a of yeast, Saccharomyces cerevisiae mutant with hmla2-102 and sir3-8ts was changed to type alpha by changing the growth temperature from 25C to 35C. A temperature-sensitive expression vector system was constructed using mating factor alpha1 (Mfalpha1) gene encoding alpha factor which is expressed in the type alpha cells. Vectors with different copy numbers were constructed by joining the promoter and pre or prepro-secretion single sequence of Mfalpha1 to promoterless PHO5' gene as a reporter gene.

• Korean Journal of Ecology and Environment
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• v.56 no.1
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• pp.1-13
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• 2023

Targeting Microcystin (MC), which is most abundantly detected in the North-Han River water area, we analyzed the relationship between the MC biosynthesis gene (mcyA gene), cyanobacteria cell density, and MC concentration, derived an RNA-MC conversion formula, and derived the cyanobacteria. The concentration of MC present in cells was predicted. In the North-Han River waters, the mcyA gene was found mainly at downstream sites of the North-Han River after Muk-Hyeon Stream junction, and higher copy numbers were found on average than other sites. In the Uiam Lake waters upstream of the North-Han River, the mcyA gene copy number increased at the Kong-Ji Stream point, and after September, the mcyA gene copy number decreased throughout the North-Han River waters. The expression of the mcyA gene was concentrated in the short period of summer due to the spatio-temporal difference between upstream and downstream water bodies. The mcyA gene expression level was not only highly correlated with MC concentration, but also correlated with the cell density of Microcystis aeruginosa and Dolichospermum circinale, which are known to biosynthesize MC. Six conversion formulas derived based on the RNA-MC relationship showed statistical significance (p<0.05) and exhibited high correlation coefficients (r) of 0.9 or higher. The expression level of MC biosynthesis gene present in eRNA determines the synthesis of cyanotoxin substances in water, quickly quantifies gene activity, and can be fully utilized for early warning of MC development.