• Journal of Korean Dental Science
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• 제16권1호
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• pp.35-46
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• 2023

Purpose: Culture-based methods for microbiological diagnosis and antibiotic susceptibility tests have limitations in the management of orofacial infections. We aimed to profile pus microbiota and identify antibiotic resistance genes (ARGs) using a culture-independent approach. Materials and Methods: Genomic DNA samples extracted from the pus specimens of two patients with orofacial abscesses were subjected to shotgun sequencing on the NovaSeq system. Taxonomic profiling and prediction of ARGs were performed directly from the metagenomic raw reads. Result: Taxonomic profiling revealed obligate anaerobic polymicrobial communities associated with infections of odontogenic origins: the microbial community of Patient 1 consisted of one predominant species (Prevotella oris 74.6%) with 27 minor species, while the sample from Patient 2 contained 3 abundant species (Porphyromonas endodontalis 33.0%; P. oris 31.6%; and Prevotella koreensis 13.4%) with five minor species. A total of 150 and 136 putative ARGs were predicted in the metagenome of each pus sample. The coverage of most predicted ARGs was less than 10%, and only the CfxA2 gene identified in Patient 1 was covered 100%. ARG analysis of the seven assembled genome/metagenome datasets of P. oris revealed that strain C735 carried the CfxA2 gene. Conclusion: A metagenomics-based approach is useful to profile predominantly anaerobic polymicrobial communities but needs further verification for reliable ARG detection.

• Journal of Biosystems Engineering
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• 제41권3호
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• pp.263-272
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• 2016

Purpose: Pulse crop damage due to wild birds is a serious problem, to the extent that the rate of damage during the period of time between seeding and the stage of cotyledon reaches 45.4% on average. This study investigated a method of fundamentally blocking birds from eating crops by conducting vinyl mulching after seeding and identifying the growing locations for beans to perform punching. Methods: Infrared (IR) sensors that could measure the temperature without contact were used to recognize the locations of soybean cotyledons below vinyl mulch. To expand the measurable range, 10 IR sensors were arranged in a linear array. A sliding mechanical device was used to reconstruct the two-dimensional spatial variance information of targets. Spatial interpolation was applied to the two-dimensional temperature distribution information measured in real time to improve the resolution of the bean coleoptile locations. The temperature distributions above the vinyl mulch for five species of soybeans over a period of six days from the appearance of the cotyledon stage were analyzed. Results: During the experimental period, cases where bean cotyledons did and did not come into contact with the bottom of the vinyl mulch were both observed, and depended on the degree of growth of the bean cotyledons. Although the locations of bean cotyledons could be estimated through temperature distribution analyses in cases where they came into contact with the bottom of the vinyl mulch, this estimation showed somewhat large errors according to the time that had passed after the cotyledon stage. The detection results were similar for similar types of crops. Thus, this method could be applied to crops with similar growth patterns. According to the results of 360 experiments that were conducted (five species of bean ${\times}$ six days ${\times}$ four speed levels ${\times}$ three repetitions), the location detection performance had an accuracy of 36.9%, and the range of location errors was 0-4.9 cm (RMSE = 3.1 cm). During a period of 3-5 days after the cotyledon stage, the location detection performance had an accuracy of 59% (RMSE = 3.9 cm). Conclusions: In the present study, to fundamentally solve the problem of damage to beans from birds in the early stage after seeding, a working method was proposed in which punching is carried out after seeding, thereby breaking away from the existing method in which seeding is carried out after punching. Methods for the accurate detection of soybean growing locations were studied to allow punching to promote the continuous growth of soybeans that had reached the cotyledon stage. Through experiments using multiple IR sensors and a sliding mechanical device, it was found that the locations of the crop could be partially identified 3-5 days after reaching the cotyledon stage regardless of the kind of pulse crop. It can be concluded that additional studies of robust detection methods considering environmental factors and factors for crop growth are necessary.

• Parasites, Hosts and Diseases
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• 제57권1호
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• pp.9-15
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• 2019

Melting temperature shift ($T_m-shift$) is a new detection method that analyze the melting curve on real-time PCR thermocycler using SYBR Green I fluorescent dye. To establish a $T_m-shift$ method for the detection of Ancylostoma ceylanicum and A. tubaeforme in cats, specific primers, with GC tail of unequal length attached to their 5' end, were designed based on 2 SNP loci (ITS101 and ITS296) of the internal transcribed spacer 1 (ITS1) sequences. The standard curve of $T_m-shift$ was established using the standard plasmids of A. ceylanicum (AceP) and A. tubaeforme (AtuP). The $T_m-shift$ method stability, sensitivity, and accuracy were tested with reference to the standard curve, and clinical fecal samples were also examined. The results demonstrated that the 2 sets of primers based on the 2 SNPs could accurately distinguish between A. ceylanicum and A. tubaeforme. The coefficient of variation (CV) of $T_m$- values of AceP and AtuP was 0.07% and 0.06% in ITS101 and was 0.06% and 0.08% in ITS296, respectively. The minimum detectable DNA concentration was $5.22{\times}10^{-6}$ and $5.28{\times}10^{-6}ng/{\mu}l$ samples of AceP and AtuP, respectively. The accuracy of $T_m-shift$ method reached 100% based on examination of 10 hookworm DNA samples with known species. In the clinical detection of hookworm in 69 stray cat fecal sample, the $T_m-shift$ detection results were consistent with the microscopic examination and successfully differentiated between the 2-hookworm species. In conclusion, the developed method is a rapid, sensitive and accurate technique and can provide a promising tool for clinical detection and epidemiological investigation of cat-derived hookworms.

• BMB Reports
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• 제37권2호
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• pp.213-222
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• 2004

A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Haliotis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N = 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CUHO3) and H. varia (CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N = 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.

• BMB Reports
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• 제37권4호
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• pp.493-502
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• 2004

The genetic diversity and species-diagnostic markers in the introduced apple snail, Pomacea canaliculata and in the native Thai apple snails; Pila ampullacea, P. angelica, P. pesmei, and P. polita, were investigated by restriction analysis of COI and are reported for the first time. Twenty-one composite haplotypes showing non-overlapping distributions among species were found. Genetic heterogeneity analysis indicated significant differences between species (P < 0.0001) and within P. pesmei (P < 0.0001) and P. angelica (P < 0.0004). No such heterogeneity was observed in Pomacea canaliculata (P > 0.0036 as modified by the Bonferroni procedure), P. ampullacea (P = 0.0824-1.000) and P. polita (P = 1.0000). A neighbor-joining tree based on genetic distance between pairs of composite haplotypes differentiated all species and indicated that P. angelica and P. pesmei are closely related phylogenetically. In addition, the 16S rDNA of these species was cloned and sequenced. A species-specific PCR for P. canaliculata was successfully developed with a sensitivity of detection of approximately 50 pg of the target DNA template. The amplification of genomic DNA (50 pg and 25 ng) isolated from the fertilized eggs, and juveniles (1, 7, and 15 d after hatching) of Pomacea canaliculata was also successful, and suggested that Pomacea canaliculata and Pila species can be discriminated from the early stages of development.

• The Plant Pathology Journal
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• 제38권6호
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• pp.656-664
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• 2022

Pectobacterium odoriferum is the primary causative agent in Kimchi cabbage soft-rot diseases. The pathogenic bacteria Pectobacterium genera are responsible for significant yield losses in crops. However, P. odoriferum shares a vast range of hosts with P. carotovorum, P. versatile, and P. brasiliense, and has similar biochemical, phenotypic, and genetic characteristics to these species. Therefore, it is essential to develop a P. odoriferumspecific diagnostic method for soft-rot disease because of the complicated diagnostic process and management as described above. Therefore, in this study, to select P. odoriferum-specific genes, species-specific genes were selected using the data of the P. odoriferum JK2.1 whole genome and similar bacterial species registered with NCBI. Thereafter, the specificity of the selected gene was tested through blast analysis. We identified novel species-specific genes to detect and quantify targeted P. odoriferum and designed specific primer sets targeting HAD family hydrolases. It was confirmed that the selected primer set formed a specific amplicon of 360 bp only in the DNA of P. odoriferum using 29 Pectobacterium species and related species. Furthermore, the population density of P. odoriferum can be estimated without genomic DNA extraction through SYBR Green-based real-time quantitative PCR using a primer set in plants. As a result, the newly developed diagnostic method enables rapid and accurate diagnosis and continuous monitoring of soft-rot disease in Kimchi cabbage without additional procedures from the plant tissue.

• Applied Biological Chemistry
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• 제46권2호
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• pp.74-78
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• 2003

Urease 활성이 중금속 이온에 의하여 저해되는 현상을 알아보기 위하여 발색시약을 사용하는 광학적인 방법으로 효소반응에 의한 암모니아 발생량을 측정하였다. 암모니아 농도에 따른 cuvette assay 시의 흡광도 중가는 암모니아 농도 3.0 mg/l 까지 직선성을 보였으며 이 때의 상관계수는 0.998(r)이었다. 중금속 이온에 의한 urease의 저해활성을 알아보기 위하여 기질에 단일 중금속 이온의 농도별 용액을 가하여 흡광도를 측정한 결과 효소의 저해도는 Hg(II)>Pb(II)>Cu(II)>Cd(II)>Zn(II) 이온의 순으로 나타났다. 한 가지의 중금속 이온 농도를 고정하고 다른 중금속 이온을 각기 다른 농도로 가하면서 urease활성을 측정하였을 때 효소의 저해도는 대체로 개별 중금속 이온에 의한 저해의 총합으로 나타났다. 상기의 결과는 본 연구의 방법이 Hg(II) 이온에 대한 선택적 검출법으로서 뿐만 아니라 여러 형태의 시료 중에 존재가능한 중금속 이온들을 정성적으로 판별하는 방법으로 활용될 수 있음을 보여주었다.

• 한국미생물·생명공학회지
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• 제36권2호
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• pp.96-100
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• 2008

recA 유전자를 특이적으로 증폭하는 PCR을 이용하여 우리나라 전통 침채류 발효에 관여하는 유산균의 다양성을 검토해 보았다. 김치에서 많이 검출되는 유산균 7종 및 대조군으로 Lactobacillus acidophilus를 검출할 수 있는 특이적 PCR primer pair을 이용하여 전통 침채류 5증(갓김치, 동치미, 배추김치, 오이소박이, 총각김치로부터 추출한 DNA를 template로 PCR을 수행한 결과, 5종의 침채류 모두에서 Lactobacillus plantarum과 Lactobacillus sakei가 검출되었지만, Lactobacillus paraplantarum, Lactobacillus pentosus와 대조군인 Lb. acidophilus는 검출되지 않았다. Lactobacillus brevis와 Leuconostoc citreum은 배추김치에서만 검출되었으며, Leuconostoc mesenteroides의 경우 갓김치, 동치미, 배추김치, 오이소박이에서 검출되었다. 주재료의 종류에 따라서 발효에 관여하는 유산균은 차이가 있는 것으로 추정되며, Lb. plantarum과 Lb. sakei가 우리나라 침채류 발효에 가장 널리 관여하는 것으로 사료된다.

• 대한수의학회지
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• 제43권4호
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• pp.677-681
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• 2003

Currently, both the indirect fluorescent antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA) have been used to detect Neospora caninum antibodies. Several factors such as the buffers, the conjugate, the pattern of fluorescence, and the cross reactivity with other apicornplexan protozoan, may result in poorly correlated data. The present study was undertaken to develop and evaluate the Neospora agglutination test (NAT) for the detection and quantification of IgG antibodies to N. caninum from various animal species. Compared to the ELISA method, the NAT with a cutoff value of 1:512 gave a high index of coincidence (kappa=0.807) and no cross reactivity to Toxoplasma gondii antiserum. Hence, this NAT method, which did not require a species-specific secondary antibody and expensive tools, would be easily available for the detection of antibodies to N. caninllm of various animal species.

• ALGAE
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• 제17권2호
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• pp.95-104
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• 2002

Population dynamics of Anabaena and the anatoxin-a concentration were monitored with physicochemical parameters at 3 sites in the lower Naktong River from May to September in 2000. Total 4 species of Anabaena (A. flosaquae, A. smithii, A. ucrainica and A. mucosa) were identified with morphological characterisitcs. Anabaena flos-aquae was most abundant among the populations. The standing crop of Anabaena ranged from 10 to 11,220 cells · $ml^{-1}$ and biomass of Anabaena more 1,000 cells · $ml^{-1}$ was obseved once at St. Mulgeum and St. Seonam, twice at St. Hagueon out of total 9 samplings. There were not significant correlations between the standing crop of Anabaena and other physicochemical parameters such as temperature, nitrate, total nitrogen, phosphate, total phophorus and N/P ratios. The frequency of trichomes with akinetes was low and ranged from 0 to 4% in the total Anabaena population and A. smithii showed highest frequency of 2.8% among all species. The population at St. Seonam showed highest frequency of 1.4% among all sampling sties. The population in September showed the highest frequency of 3.0% among all sampling period. The frequency of trichomes with heterocysts was low and ranged from 1 to 87% inthe total Anabaena population and A. smithii showed highest frequency of 55.1% among all species. The population at St. Mulgeum showed highest frequency of 17.6% among all sampling sites. The population in August showed the highest frequency of 21.4% among all sampling period. The frequency of trichomes with akinetes and/or heterocysts was not related to all the physicochemical parameters of temperature, nitrate, total nitrogen, phosphate, total phosphorus and N/P ratios. The anatoxin-a concentations were determined in algal materials dominated by Microcystis and Anabaena from June though August by derivatization using 7-fluoro-4-nitro-2, 1,3-benzoxadiazole (NBD-F) and HPLC analysis with fluorimetric detection. All the concentrations were below the detection limit of 0.1 ㎍ · $l^{-1}$ in the present study.