• Title/Summary/Keyword: Species-specific PCR

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First report of the photosynthetic dinoflagellate Heterocapsa minima in the Pacific Ocean: morphological and genetic characterizations and the nationwide distribution in Korea

  • Lee, Sung Yeon;Jeong, Hae Jin;Kwon, Ji Eun;You, Ji Hyun;Kim, So Jin;Ok, Jin Hee;Kang, Hee Chang;Park, Jae Yeon
    • ALGAE
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    • v.34 no.1
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    • pp.7-21
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    • 2019
  • The genus Heterocapsa is one of the major dinoflagellate groups, with some of its species having worldwide distributions. However, prior to the present study, the phototrophic species Heterocapsa minima has been reported only from the northeast Atlantic Ocean. Recently, H. minima was found in the Korean waters, and a clonal culture was established. This culture was used to examine the morphology of the Korean strain H. minima HMMJ1604 through light and scanning electron microscopy, as well as for its genetic characterization. Furthermore, to determine the nationwide distribution of H. minima in Korea, its abundance was quantified in the waters of 28 stations in all four seasons in 2016-2018 using the quantitative real-time polymerase chain reaction method. The overall morphology of H. minima HMMJ1604 was very similar to that of the Irish strain H. minima JK2. However, the Korean strain had five pores around the pore plate, whereas the Irish strain had six pores. When properly aligned, the sequences of the large subunit and internal transcribed spacer regions of the ribosomal DNA of the Korean strain were identical to those of the Irish strain. This species was detected in the waters of 26 out of 28 stations, but its abundance was greater than $1.0cells\;mL^{-1}$ at 8 stations. The highest abundance of H. minima was $44.4cells\;mL^{-1}$. Although this species was found in all seasons, its abundance was greater than $1.0cells\;mL^{-1}$ when the water temperature and salinity were $10.9-25.0^{\circ}C$ and 17.5-34.1, respectively. To the best knowledge, the present study reported for the first time that H. minima lives in the Pacific Ocean and is widely distributed in the Korean waters.

Monitoring Culicine Mosquitoes (Diptera: Culicidae) as a Vector of Flavivirus in Incheon Metropolitan City and Hwaseong-Si, Gyeonggi-Do, Korea, during 2019

  • Bahk, Young Yil;Park, Seo Hye;Kim-Jeon, Myung-Deok;Oh, Sung-Suck;Jung, Haneul;Jun, Hojong;Kim, Kyung-Ae;Park, Jong Myong;Ahn, Seong Kyu;Lee, Jinyoung;Choi, Eun-Jeong;Moon, Bag-Sou;Gong, Young Woo;Kwon, Mun Ju;Kim, Tong-Soo
    • Parasites, Hosts and Diseases
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    • v.58 no.5
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    • pp.551-558
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    • 2020
  • The flaviviruses are small single-stranded RNA viruses that are typically transmitted by mosquitoes or tick vectors and are etiological agents of acute zoonotic infections. The viruses are found around the world and account for significant cases of human diseases. We investigated population of culicine mosquitoes in central region of Korean Peninsula, Incheon Metropolitan City and Hwaseong-si. Aedes vexans nipponii was the most frequently collected mosquitoes (56.5%), followed by Ochlerotatus dorsalis (23.6%), Anopheles spp. (10.9%), and Culex pipiens complex (5.9%). In rural regions of Hwaseong, Aedes vexans nipponii was the highest population (62.9%), followed by Ochlerotatus dorsalis (23.9%) and Anopheles spp. (12.0%). In another rural region of Incheon (habitat of migratory birds), Culex pipiens complex was the highest population (31.4%), followed by Ochlerotatus dorsalis (30.5%), and Aedes vexans vexans (27.5%). Culex pipiens complex was the predominant species in the urban region (84.7%). Culicine mosquitoes were identified at the species level, pooled up to 30 mosquitoes each, and tested for flaviviral RNA using the SYBR Green-based RT-PCR and confirmed by cDNA sequencing. Three of the assayed 2,683 pools (989 pools without Anopheles spp.) were positive for Culex flaviviruses, an insect-specific virus, from Culex pipiens pallens collected at the habitats for migratory birds in Incheon. The maximum likelihood estimation (the estimated number) for Culex pipiens pallens positive for Culex flavivirus was 25. Although viruses responsible for mosquito-borne diseases were not identified, we encourage intensified monitoring and long-term surveillance of both vector and viruses in the interest of global public health.

Application of Multiplex RT-PCR for Simultaneous Identification of Tomato Spotted Wilt Virus and Thrips Species in an Individual Thrips on Chrysanthemum (시설재배 국화에서 총채벌레의 종 동정 및 보독 바이러스 동시 검출을 위한 다중 진단법 적용)

  • Yoon, Ju-Yeon;Yoon, Jung-Beom;Seo, Mi-Hye;Choi, Seung-Kook;Cho, In-Sook;Chung, Bong-Nam;Yang, Chang Yeol;Gangireddygari, Venkata Subba Reddy
    • Research in Plant Disease
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    • v.26 no.4
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    • pp.264-271
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    • 2020
  • We have developed a simultaneous diagnostic method that can identify both the species of thrips and tomato spotted wilt virus (TSWV) that are problematic in chrysanthemum plants. This is a method of amplifying DNA by performing reverse transcription-polymerase chain reaction by simultaneously adding primers specific to TSWV coat protein (N) gene and primers specific to the internal transcribed spacer 2 region of Frankliniella occidentalis and F. intonsa using total nucleic acid extracted from one thrips. The sizes of DNA fragments for TSWV, F. occidentalis, and F. intonsa were 777, 287, and 367 bp, respectively. These results showed species identification of thrips and whether thrips carrying TSWV can be simultaneously confirmed. Further usefulness of the simultaneous diagnostic method was made from greenhouse survey at chrysanthemum greenhouses in Taean (Chungcheongnam-do) and Changwon (Gyeongsangnam-do) to investigate the identification of thrips species and the rate of thrips carrying TSWV. Of thrips collected from the greenhouses, 83.7% thrips was F. occidentalis and 72.9% F. occidentalis carried TSWV in Taean. Similarly, the diagnostic method showed that 92.2% thrips was F. occidentalis and 84.0% F. occidentalis carried TSWV in Changwon. These results confirm that F. occidentalis is a dominant thrips species and the thrips species plays a crucial role in the transmission of TSWV in chrysanthemum plants in the greenhouses. Taken together, this study showed a simple diagnostic method for thrips identification and epidemiological studies of the timing and spread of TSWV through thrips in chrysanthemum greenhouses in South Korea.

Spread of Cyst Nematodes in Highland Chinese Cabbage Field in Gangwon-do (강원도 고랭지배추 재배지에서 씨스트선충의 분포 확산)

  • Kwon, Soon-Bae;Park, Dong-Kwon;Won, Heon-Seop;Moon, Youn-Gi;Lee, Jae-Hong;Kim, Yong-Bog;Choi, Byoung-Gon;Seo, Hyun-Taek;Ko, Hyoung-Rai;Lee, Jae-Kook;Lee, Dong Woon
    • Korean journal of applied entomology
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    • v.57 no.4
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    • pp.339-345
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    • 2018
  • The sugar beet cyst nematode (SBCN), Heterodera schachtii first detected in Taebaek, Gangwon-do in 2011, is one of the major plant parasitic nematodes that cause economic damage to the Chinese cabbage in highland regions. In addition, the distribution of clover cyst nematode (CCN), H. trifolii was confirmed in the highland Chinese cabbage cultivated regions in 2017. In order to investigate the spread of cyst nematodes, this study has been conducted since 2013 in the highland Chinese cabbage cultivation area. In addition, in 2017, the Real-Time PCR technique with the species-specific primer was used to investigate those two cyst nematodes and the soybean cyst nematode (SCN), H. glycines which is known for its distribution in Korea, focusing on the main production regions of highland Chinese cabbage cultivation. The number of infected fields in the Chinese cabbage plantation in highland increased every year to confirm distribution in Taebaek, Samcheok, Jeongseon and Gangneung in 2017, and the cumulative number of infection fields reached 245 by 2017. Of the 41 possible cyst nematode samples for PCR analysis, 61% were CCN, only 9.8% of the SBCN and 29.3% of the SCN were identified. Therefore, some of the previously known SBCN or CCN discoveries are likely to have been infected with SCN. It is believed that the CCN needs to be controlled in the future as CCN have been found to be dominant species in the highland Chinese cabbage plantation regions.

Detection of Toxoplasma antigens and antibodies in mice infected with different strains of Toxoplasma gonnii (톡소포자충의 충주에 따른 항원과 항체의 검출 시기 및 양상)

  • 이영하;김재영
    • Parasites, Hosts and Diseases
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    • v.33 no.3
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    • pp.201-210
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    • 1995
  • This study aims to assess the possible strain-dependent variations in detection of ToxopLosmn antigens and antibodies. The virulent RH strain or avirulent Beverley strain of T gondii were injected into mice, intraperitoneally, and their antigens, antibodies and parasites were identified from the blood or tissues: liver, brain and spleen by ELISA, Western blot and PCR. In mice infected with RH strain, circulating antigens and parasitemia were first detected from 2 days after infection, and ToxopIasma DNA were found in the blood, liver, brain and spleen from 3 days after infection. It was impossible to detect specific IgM and IgG antibodies to T gondij and any specific band was not found by Western blot. In mice infected with Beverley strain, circulating antigens were detected between day 10 and day 35. The Toxoplusma DNA was found in the blood and liver from day 15 until day 60, and in the brain from day 20. But Toxoplosma DNA in the spleen were mainly detected between day 10 and day 30. The IgM antibodies were first appeared on day 10 post-infection, and were noted obviously increased between day 15 and 25. The IgG antibodies were first detected on day 15, and showed progressively increased titers. The antibody binding bands were specific according to infection period. Sera from mice infected with Beverley strain reacted mainly with the antigen of 27.5-kDa and 32.5-kDa. In conclusion, mice infected with RH strain revealed Toxoplosma antigens strongly, but not antibodies. However. mice infected with Beverley strain revealed both the Toxoplasma antigens and antibodies. The present results showed that immune responses are different between avirulent and virulent T gonnii.

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Ttrosine Hydroxylase in Japanese Medaka (Oryzias latipes): cDNA Cloning and Molecular Monitoring of TH Gene Expression As a Biomarker (송사리 Tyrosine Hydroxylase: cDNA 클로닝 및 생물지표로서의 TH 유전자 발현의 분자생물학적 추적)

  • Shin, Sung-Woo;Kim, Jung-Sang;Chon, Tae-Soo;Lee, Sung-Kyu;Koh, Sung-Cheol
    • Environmental Analysis Health and Toxicology
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    • v.15 no.4
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    • pp.131-137
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    • 2000
  • The release of hazardous waste materials into the environment poses serious risks in humans and ecosystems. The risk assessment of environmental pollutants including hazardous chemicals requires a comprehensive measurement of hazard and exposure of the chemicals that can be achieved by toxicity evaluation using a biological system such as biomarkers. In this report we have tried to develop a biomarker used to elucidate a molecular basis of, and to monitor abnormal behaviors caused by diazinon in Japanese medaka (Oryzias latipes) as a model organism. First, an attempt was made to clone tyrosine hydroxylase gene from Japanese medaka that would be a candidate for a biomarker for neuronal modulations and behaviors. For monitoring experiments at behavioral and molecular biological levels, the fish were treated under different sublethal conditions of diazinon and their behavioral responses were observed . In this study we have successfully cloned a partial TH gene from the medaka fish through PCR screening of an ovary cDNA library. DNA sequencing analysis revealed that the amplified fragment was 327 bp encoding 109 amino acids. Comparing the DNA sequence of medaka TH with other species, TH gene revealed the DNA sequence was completely identical to that of rat TH. In the RT-PCR, 330 Up of mRNA was consistently amplified in all the treated samples including control There were no significant differences in the TH expression level regardless of treating concentrations (1∼5,000 ppb) and time (0∼48 hr) The reason appeared to be that RT-PCR was not performed using through a quantitative analysis normalized against an actin gene expression. Organ or tissue - specific detection of TH activity and mRNA as biomarkers will be a useful monitoring tool for neurobehavioral changes in fish influenced by toxic chemicals. Furthermore, quantitative analysis of locomotive patterns and its correlation with the neurochemical and molecular data would be highly useful in measuring toxicity and hazard ofvarious environmental pollutants.

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The Prevalence of Oral Spirochetes in Korean Adult Periodontitis (한국인 성인성 치주염 환자에서의 구강 스피로헤타의 분포)

  • Kim, Hay-Hyun;Choi, Bong-Kiu;Choi, Seong-Ho;Chai, Jung-Kiu;Kim, Chong-Kwan;Cho, Kyoo-Sung
    • Journal of Periodontal and Implant Science
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    • v.28 no.4
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    • pp.659-678
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    • 1998
  • In the present study, oligonucleotide probes based on 16S rRNA were taken to investigate the diversity of oral spirochetes without culture method. This is the first study that revealed oral spirochetes of both presently cultivable and uncultured oral spirochetes in Korean adult periodontitis patients. Subgingival plaque samples were taken from diseased sites(probing depth ${\geq}6\;mm$, experimental group, n=116) and healthy sites(probing depth${\leq}3mm$, control 1 group, n=28) in 29 patients with adult periodontitis, and from 20 periodontally healthy subjects(probing depth${\leq}3mm$, control 2 group, n=100). Following being examined under phase-contrast microscope, all samples were submitted to dot-blot hybridization after polymerase chain reacton with eubacterial primers. 5 species-specific probes(TVIN, TDEN, TMAL, TSOC, and TPEC) and 7 group-specific probes(TRE I, TRE II, TRE III, TRE IV, TRE V, TRE VI, and TRE VII) were used one by one for the identification of both cultivable and so far uncultivable oral spirochetes. All probes were labeled with digoxigenin(DIG)-ddUTP and detected by chemilumininescence. The following results were obtained. 1. Under phase-contrast microscope, 91.37% and 14.28% of oral spirochetes were observed in the experimental and control 1 groups, respectively. None of oral spirochetes were observed in control 2 group. 2. With universal probe, 98.27%, 46.42%, and 22.0% of oral spirochetes were observed in experimental, control 1, and control 2 groups, respectively. 3. With specific probe, 95.68%, 35.71%, and 19.0% of oral spirochetes were observed in experimental, control 1, and control 2 groups, respectively. 4. With species-specific probes, T. socranskii were recovered in a high percentage of sites(81.89%) examined, followed by T. maltophilum(50.0%), T. vincentii(36.20%), T. denticola(13.79%), respectively. With group- specific probes, TRE IV was recovered in a high percentage of sites(85.34%) examined, followed by TRE II(77.58%), TRE I(56.89%), TRE III(25.86%), TRE VI(5.17%), and TRE V(2.58%), respectively. 5. T. vincentii were only observed in the diseased sites, not in the healthy sites. 6. Neither T. pectinovorum nor group VII oral spirochetes were observed in any sites. The findings warrant further investgations of the recovered spirochetes to elucidate the possible associations of oral spirochetal prevalence in race and types of periodontitis, pathogenesis of T. vincentii and the possible distributional change of oral spirochetes before and after treatments.

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The Brassica rapa Tissue-specific EST Database (배추의 조직 특이적 발현유전자 데이터베이스)

  • Yu, Hee-Ju;Park, Sin-Gi;Oh, Mi-Jin;Hwang, Hyun-Ju;Kim, Nam-Shin;Chung, Hee;Sohn, Seong-Han;Park, Beom-Seok;Mun, Jeong-Hwan
    • Horticultural Science & Technology
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    • v.29 no.6
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    • pp.633-640
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    • 2011
  • Brassica rapa is an A genome model species for Brassica crop genetics, genomics, and breeding. With the completion of sequencing the B. rapa genome, functional analysis of the genome is forthcoming issue. The expressed sequence tags are fundamental resources supporting annotation and functional analysis of the genome including identification of tissue-specific genes and promoters. As of July 2011, 147,217 ESTs from 39 cDNA libraries of B. rapa are reported in the public database. However, little information can be retrieved from the sequences due to lack of organized databases. To leverage the sequence information and to maximize the use of publicly-available EST collections, the Brassica rapa tissue-specific EST database (BrTED) is developed. BrTED includes sequence information of 23,962 unigenes assembled by StackPack program. The unigene set is used as a query unit for various analyses such as BLAST against TAIR gene model, functional annotation using MIPS and UniProt, gene ontology analysis, and prediction of tissue-specific unigene sets based on statistics test. The database is composed of two main units, EST sequence processing and information retrieving unit and tissue-specific expression profile analysis unit. Information and data in both units are tightly inter-connected to each other using a web based browsing system. RT-PCR evaluation of 29 selected unigene sets successfully amplified amplicons from the target tissues of B. rapa. BrTED provided here allows the user to identify and analyze the expression of genes of interest and aid efforts to interpret the B. rapa genome through functional genomics. In addition, it can be used as a public resource in providing reference information to study the genus Brassica and other closely related crop crucifer plants.

Detection of Pseudomonas tolaasii Causing Brown Blotch Disease of Mushroom with Species-specific DNA Probe (종 특이 DNA probe를 이용한 버섯 세균성 갈반병 병원균(Pseudomonas tolaasii)의 검출)

  • Kwon, Soon-Wo;Go, Seung-Joo;Cheun, Meung-Sook;Kang, Hee-Wan;Oh, Se-Jong;Chang, Who-Bong;Ryu, Jin-Chang
    • The Korean Journal of Mycology
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    • v.27 no.2 s.89
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    • pp.132-137
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    • 1999
  • This study was carried out to develop the molecular marker for the detection of Pseudomonas tolaasii, a causative agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). When several primers designed from repetitive sequences and pectin lyase genes of bacteria were used to produce DNA polymorphism from different Pseudomonas spp. isolated from edible mushrooms, PEU1 primer derived from pectin lyase gene produced polymorphic bands differentiating P. tolaasii strains from other Pseudomonas species. Two bands, 1.0kb and 0.4kb, found commonly in 6 isolates of P. tolaasii were cloned into pGEM-T vector which were designated as pPTOP1 and pPTOP2, respectively, to use as probe. The 0.4 kb insert of pPTOP2 hybridized to only 6 isolates of P. tolaasii, but did not to the other Pseudomonas species. As few as $1.5{\times}10^3$ colony forming unit (cfu) of P. tolaasii could be detected by dot blot hybridization with the cloned 0.4kb DNA in pPTOP2.

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Genetic Differences within and between Populations of Korean Catfish (S. asotus) and Bullhead (P. fulvidraco) Analysed by RAPD-PCR

  • Yoon, Jong-Man;Kim, Jong-Yeon
    • Asian-Australasian Journal of Animal Sciences
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    • v.17 no.8
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    • pp.1053-1061
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    • 2004
  • Of the 20 arbitrarily chosen primers, six oligonucleotides decamer primers were used on the basis of the number of the polymorphisms generated in catfish (Silurus asotus) from Yesan and bullhead (Pseudobagrus fulvidraco) from Dangjin in Korea. Six primers were used generating a total of 602 scorable bands in catfish and 195 in bullhead population, respectively, ranging in size of DNA fragments from less than approximately 100 to larger than 2,000 base pairs (bp). Six primers yielded 199 polymorphic fragments (33.1%) in catfish and 47 (24%) in bullhead, respectively. In the present study, a total of 328 common fragments (an average of 54.7 per primer) were observed in catfish population, whereas 84 (an average of 14.0 per primer) in bullhead. The total number of specific fragments in catfish and bullhead population were 76 and 64, respectively. In catfish population, random decamer, OPA-17 (GACCGCTTGT) generated the highest number of fragments (a total of 141) in comparison with other primers used, with an average of 11.8. The common bands in the molecular weight of 300 bp generated by random primer OPA-06 (GGTCCCTGAC) were present in every individuals in bullhead population. The major polymorphic bands in the molecular weight of 100 bp generated by OPA-17 were identified in lane 14, 15, 17, 18, 19 20 and 21, which were identifying species in bullhead population. The average bandsharing values (BS values) of all of the samples within catfish population ranged from 0.575 to 0.945, whereas 0.063-1.000 within bullhead population. The bandsharing value (index of similarity between individuals) between individual No. 5 and No. 9 showed the highest level within catfish population, whereas the bandsharing value between individual No. 1 and No. 2 showed the lowest level. The single linkage cluster analysis resulted from four primers, indicating four genetic groupings composed of group 1 (C1-C10, all of the catfish samples), group 2 (B11, B12, B13, B14, B16, B17, B18, B19), group 3 (B15) and group 4 (B20 and B21). The dendrogram reveals close relationships between individual identities within two species populations and individuals derived from the same ancestor, respectively. However, genetic distances between two species populations ranged from 0.124 to 0.333. The shortest genetic distance (0.042) displaying significant molecular differences was between individual No. 6 and No. 9 catfish population. The shortest genetic distance (0.033) displaying significant molecular differences also was between individual No. 18 and No. 19 in bullhead population. Reversely, the genetic distance of individual No. 20/21 among individuals in bullhead population was highest (0.333). This result showed that bullhead No. 20 and 21 were distinct from other individuals within bullhead population.