• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1401-1409
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• 2015

The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 10⁰ fg/µl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.

• 한국미생물·생명공학회지
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• 제46권2호
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• pp.171-179
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• 2018

Human papillomavirus (HPV) plays a critical role in the development of cervical carcinoma. This study analyzed the efficiency of multiplex real-time PCR in detecting and identifying HPV genotypes in samples from women who visited a Korean hospital for checkups. Cervical swab specimens were obtained from women who attended a checkup at the Health Improvement Center of Hospital in Dankook University Cheonan, South Korea and were referred for an HPV genotyping test between January and September 2014. A total of 1703 cervical swab specimens were collected consecutively during this period. PCR results were compared with those of the traditional cytological assay for the same population. Among the 1,703 specimens, 19.91% were HPV positive, of which 14.50% indicated a single infection and 5.40% indicated multiple infections. However, cytology identified only 2.52% of positive cases, including 1.23% cases of atypical squamous cells of undetermined significance, 1% of low grade squamous intra-epithelial lesion, and 0.29% of high grade squamous intra-epithelial lesion. The rate of high-risk and low-risk HPV in the abnormal cytology group was 48 and 23, respectively, and 274 and 136 in the normal group, respectively. HPV types 56, 52, 43 were the most prevalent in that order. Our results confirm the efficiency of the HPV DNA assay for the detection of 28 different HPV genotypes with reasonable sensitivity. A screening strategy that comprises the HPV DNA assay and cytology would help overcome the low sensitivity of a cytological diagnosis.

• Mycobiology
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• 제30권4호
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• pp.197-201
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• 2002

Specific primer sets based on ribosomal DNA(rDNA) internal transcribed specer(ITS) sequences were designed for rapid detection of Phellinus linteus and P. baumii. Polymerase chain reaction(PCR) with these primers produced unique bands for each Phellinus species. The annealing temperature range is from $40^{\circ}C\;to\;55^{\circ}C$. The length of PCR products(P. linteus and P. baumii) using designed combinative primer sets of PL1F, PL2R, PB1F, PB2R, ITS5F and ITS4R, were from 520 by to 730 bp. Fifteen strains of Phellinus species including P. linteus, P. baumii, P. weirianus, P. johnsonianus, P. rhabarberinus, P. pini, P. gilvus, P. igniarius, P. nigricans and P. laevigatus were examined in this study. Five strains, including two isolated strains of P. linteus(MPNU 7001 and MPNU 7002), and two isolated strains of P. baumii(MPNU 7004 and MPNU 7005) were shown to have about 520 bp (PL1F-PL2R), 700 bp (TTS5F-PL2R) and 600 bp (PB1F-ITS4R) -sized PCR single bands respectively. This molecular genetic technique provided a useful method for rapid detection and identification of P. linteus and P. baumii.

• 대한수의학회지
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• 제57권2호
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• pp.79-88
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• 2017

The study of canine vector-borne diseases in the Philippines started in the 1970s but only gained interest in the past decade. Characterization of such diseases in the Philippines remains incomplete, thus, it is necessary to obtain additional information on the prevalence and diversity of canine tick-borne diseases in the country. In this study, blood samples were obtained at two veterinary clinics in Metro Manila, Philippines from 114 dogs suspected of having canine tick-borne pathogens. Polymerase chain reaction (PCR) was performed on whole blood DNA extracts followed by sequencing, and the following pathogens were detected: Hepatozoon (H.) canis (5.26%), Babesia (B.) vogeli (5.26%), Ehrlichia (E.) canis (4.39%), and Anaplasma platys (3.51%). Additionally, a set of multiplex PCR primers were developed to detect H. canis, Babesia spp. (B. canis and B. vogeli), and E. canis in canine blood. Multiplex and conventional single-reaction PCR results for the 114 dog blood samples were similar, except for one H. canis sample. Multiplex PCR is, therefore, a useful tool in screening infected dogs in veterinary clinics. This study's results, together with those of previous studies in the country, show that canine vector-borne pathogens are an emerging veterinary concern in the Philippines.

• Asian-Australasian Journal of Animal Sciences
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• 제27권10호
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• pp.1411-1416
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• 2014

Gender selection is important in livestock industries; for example, female calves are required in the dairy industry. Sex-sorted semen is commonly used for the production of calves of the desired gender. However, assessment of the sex ratio of the sorted semen is tedious and expensive. In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay. In this assay, the X and Y chromosome-specific markers, i.e., bovine proteolipid protein (PLP) gene and sex-determining region Y (SRY) were simultaneously quantified in a single tube. The multiplex real-time PCR assay was shown to have high amplification efficiencies (97% to 99%) comparable to the separated-tube simplex real-time PCR assay. The results obtained from both assays were not significantly different (p>0.05). The multiplex assay was validated using reference DNA of known X ratio (10%, 50%, and 90%) as templates. The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen. The multiplex real-time PCR assay as shown in this study can thus be used to assess purity of sex-sorted semen.

• 한국동물위생학회지
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• 제28권3호
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• pp.259-265
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• 2005

The melanocortin 1 receptor (MC1R) encoded by the coat color extension gene (E) plays a key role in the signaling pathway of melanin synthesis. The primers for the amplification of bovine MC1R gene were designed based on a bovine MC1R gene sequence (GenBank accession no. Y19103). A size of 483bp (482bp for Hanwoo) was amplified by PCR, digested with Hpa II restriction enzyme and electrophoresed in $1.5\%$ agarose gel. When the amplified DNA product (483 bp) was digested with Hpa II restriction enzyme, Hanwoo meat showed a single band of 482bp, whereas two fragments of 325bp and 158bp were detected in Holstein, Angus and meat of Hanwoo / Holstein cross cow having back coat color phenotype, respectively. The results of this experiment Indicate that new designed primers of bovine MCIR gene may be useful for identification of Hanwoo meat from Holstein, Black Angus and Hanwoo / Holstein cross cow meat.

• 한국동물위생학회지
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• 제37권3호
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• pp.213-218
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• 2014

In early April 2014, a month-old Alexandrine Paraqeet (Psittacula eupatria) that was raised in a domestic aviary located in Gyungju-si, Korea was suddenly died and submitted to Animal Disease Intervention Center, Kyungpook National University in order to diagnose the causative agent. In post-mortem examination, the bird had abnormally developed feathers on the neck and abdomen region and subcutaneous hemorrhages on the neck and cheek adjacent to the beak. At necropsy, the bird had hemorrhage on the muscle of the femoral region, ascites, multi-focal hemorrhages on the epicardium, and diffuse hemorrhages on the sub-serosa of proventriculus and gizzard, suggesting typical avian polyomavirus (APV) infection. The partial large tumor (T) antigen gene of APV was detected by PCR from tissues of the heart, lung, liver, kidney, proventriculus and feathers of the APV-suspected birds. However, other pathogenic virus-specific nucleic acid common with psittacine birds such as avian bornavirus, psittacine beak and feather disease virus and psittacid herpesvirus were not detected from the mixed tissue samples of the bird, indicating this case is due to single infection of APV. Nucleotide sequence analysis of the partially amplified large T antigen DNA was confirmed to have 99~100% homology with that of the previously reported APV strains. This case report describes the first detection of APV in Alexandrine Paraqeet in Korea.

• Animal cells and systems
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• 제5권3호
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• pp.237-241
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• 2001

Three STR loci (STRX1[AGAT]$_{n}$, HPRTB[AGAT]$_{n}$ and ARA[AGC]$_{n}$) on X chromosome and three other STR loci (DYS390[CTG(A)T]$_{n}$, DYS392[ATT]$_{n}$ and DYS39［GATA]$_{n}$) on Y chromosome were analyzed in 154 unrelated healthy Korean subjects. Four loci (STRX1, HPRTB, DYS390 and DYS393) were amplified by quadruplex polymerase chain reaction (PCR) using fluorescent labeled primers (FLP). ARA and DYS392 were amplified separately using single PCR, similarly by using FLP. They were then run in an automated DNA sequencer and were analyzed with Genescan software. We found 7 alleles (308-332 bp) in STRX1, 7 alleles (275-299 bp) in HPRTB, 16 alleles (252-315 bp) in ARA, 6 alleles (203-223 bp) in DYS390, 7 alleles (245-263bp) in DYS392 and 5 alleles (116-132 bp) in DYS393. The ＊13(34%), ＊13(5l%), ＊23 (l8%), ＊23(50%), ＊14(39%) and ＊13(40%) alleles were observed to be the highest frequencies of STRX1, HPRTB, ARA, DYS390, DYS392 and DYS393, respectively. The detection of STR loci on sex chromosomes by quadruplex PCR allows simple determination of sex and identification of an individual. individual.

• 한국자원식물학회지
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• 제28권3호
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• pp.341-349
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• 2015

Magnoliae Flos (Sini in Korean) is one of the most important oriental medicinal plants. In the Korean Herbal Pharmacopeia, the bud of the all species in Manolia denudate and Manolia genus were regarded as the botanical sources for ‘Sini’. Most the dried bud of Manolia denudata, Manolia biondii and Manolia sprengeri were used as ‘Xin-yi’ in China. Therefore, the purpose of this study was to determine and compare the ‘Magnolia’ species, four species including Manolia denudata, M. biondii, M. liliiflora and M. Kobus were analysis of sequencing data revealed DNA polymorphisms. The based on tRNA coding leucine/phenylalanine (trnL-F) and NADH-plastoquinone oxidoreductase subunit 5 (ndhF) sequences in chloroplast DNA. For the identification of ‘Magnolia’ species, polymerase chain reaction (PCR) analysis of chloroplast DNA regions such as ndhF have proven an appropriate method. A single nucleotide polymorphism (SNP) has been identified between genuine “Sini” and their fraudulent and misuse. Specific PCR primers were designed from this polymorphic site within the sequence data, and were used to detect true plants via multiplex PCR.

• Bulletin of the Korean Chemical Society
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• 제34권4호
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• pp.1137-1144
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• 2013

$TiO_2$ composites with seven different carbon materials (activated carbons, graphite, carbon fibers, single-walled carbon nanotubes, multi-walled carbon nanotubes, graphene oxides, and reduced graphene oxides) that are virgin or treated with nitric acid are prepared through an evaporation method. The photocatalytic activities of the as-prepared samples are evaluated in terms of $H_2$ production from aqueous methanol solution (photo-catalytic reduction: PCR) and degradation of aqueous pollutants (phenol, methylene blue, and rhodamine B) (photocatalytic oxidation: PCO) under AM 1.5-light irradiation. Despite varying effects depending on the kinds of carbon materials and their surface treatment, composites typically show enhanced PCR activity with maximum 50 times higher $H_2$ production as compared to bare $TiO_2$. Conversely, the carbon-induced synergy effects on PCO activities are insignificant for all three substrates. Colorimetric quantification of hydroxyl radicals supports the absence of carbon effects. However, platinum deposition on the binary composites displays the enhanced effect on both PCR and PCO reactions. These differing effects of carbon materials on PCR and PCO reactions of $TiO_2$ are discussed in terms of physicochemical properties of carbon materials, coupling states of $TiO_2$/carbon composites, interfacial charge transfers. Various surface characterizations of composites (UV-Vis diffuse reflectance, SEM, FTIR, surface area, electrical conductivity, and photoluminescence) are performed to gain insight on their photocatalytic redox behaviors.