• Title/Summary/Keyword: PCR detection

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Development of Detection Method for Niphon spinosus, Epinephelus bruneus, and Epinephelus septemfasciatus using 16S rRNA Gene (16S rRNA를 이용한 다금바리, 자바리, 능성어 판별법 개발)

  • Park, Yong-Chjun;Jung, Yong-Hyun;Kim, Mi-Ra;Shin, Joon-Ho;Kim, Kyu-Heon;Lee, Jae-Hwang;Cho, Tae-Yong;Lee, Hwa-Jung;Lee, Sang-Jae;Han, Sang-Bae
    • Korean Journal of Food Science and Technology
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    • v.45 no.1
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    • pp.1-7
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    • 2013
  • Niphon spinosus, Epinephelus bruneus, and Epinephelus septemfasciatus are involved in the Perciformes Order and Serranidae Family. When E. bruneus and E. septemfasciatus are fully grown, the striped pattern on the body gradually disappears. Therefore, morphological classification of adult fishes is quite difficult to identify the differences to N. spinosus. In this study, we investigate the method to differentiate those using PCR. To design the primers, 16S rRNA region of N. spinosus, E. bruneus, and E. septemfasciatus registered in the GeneBank (www.ncbi.nlm.nih.gov) have been used and for the analysis, Bio Edit ver. 7.0.9.0 was used. As a result, it was design NS-003-F/NS-005-R (136 bp), EB-001-F/EB-002-R (181 bp), and ES-001-F/ES-001-R (123 bp) primers for the differentiation of each 3 different fishes. Therefore, the species-specific primer sets would be a useful tool for scientific and speedy differentiation against the illegal distribution for consumer protection.

Occurrence and eradication of Plum pox virus on Ornamentals in Korea, 2016-2017 (2016-2017년 국내 핵과류에서의 자두곰보병 발생 및 방제)

  • Kim, Mikyeong;Kim, Gi-Su;Kwak, Hae-Ryun;Kim, Jeong-Eun;Seo, Jang-Kyun;Hong, Seong-Jun;Lee, Gyeong-Jae;Kim, Ju-Hui;Choi, Min-Kyeong;Kim, Byeong-Ryeon;Kim, Ji-Gwang;Han, In-Yeong;Lee, Hyeon-Ju;Won, Heon-Seop;Kang, Hyo-Jung;Han, Jong-Woo;Ko, Suk-Ju;Kim, Hyo-Jeong;Kim, Seung-Han;Lee, Jung-Hywan;Choi, Hong-Soo
    • Research in Plant Disease
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    • v.25 no.1
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    • pp.8-15
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    • 2019
  • Plum pox virus (PPV) is a significant viral disease in Prunus spp. worldwide. A nationwide survey was started in Prunus spp. orchards, since PPV was first detected from peach in Korea, 2015. During 2016-2017, samples were collected from 30,333 trees in 1,985 orchards of stone fruits in 8 provinces and 4 cities, Korea and tested by RT-PCR using specific PPV primer set. As a result, 21 trees including peach (9 trees), Japanese apricot (4 trees), plum (1 tree), apricot (7 trees) in 10 orchards were infected and controlled by eradication program. Amplicons of the expected size (547 bp) were obtained from total RNA of seven peach trees in 2016, and directly sequenced. BLAST analysis revealed the highest nucleotide (NT) identity (99%) with a PPV D isolates (LC331298, LT600782) in Genbank. The seven isolates from shared nt sequence identities of 98 to 100% with one another. Phylogenetic analysis showed the isolates in peach clustered closely with the PPV-D isolates from Korea, Japan, USA, and Canada. This is, to our knowledge, the first report of the presence of PPV in Prunus spp. orchards in Korea, 2016-2017, we hope that our results and efforts will contribute to effective measures for eradication of PPV.

Research status of the development of genetically modified papaya (Carica papaya L.) and its biosafety assessment (GM 파파야 개발 및 생물안전성 평가 연구 동향)

  • Kim, Ho Bang;Lee, Yi;Kim, Chang-Gi
    • Journal of Plant Biotechnology
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    • v.45 no.3
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    • pp.171-182
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    • 2018
  • Papaya (Carica papaya L.) is one of the crops widely planted in tropical and subtropical areas. The papaya fruit has low calories and are plentiful in vitamins A and C and in minerals. A major problem in papaya production is a plant disease caused by the papaya ringspot virus (PRSV). The first PRSV-resistant GM papaya expressing a PRSV coat protein gene was developed by USA scientists in 1992. The first commercial GM papaya cultivars derived from the event was approved by the US government in 1997. Development of transgenic papayas has been focused on vaccine production and limited agricultural traits, including insect and pathogen resistance, long shelf life, and aluminum and herbicide tolerance. Approximately 17 countries, including the USA and China, produced transgenic papayas and/or commercialized them, which provoked studies on biosafety assessment and development of GM-detection technologies. For the biosafety assessment of potential effects on human health, effects of long-term feeding to model animals have been studied in terms of toxicity and allergenicity. Studies on environmental safety assessment include influence on soil-microbial biodiversity and transfer to soil bacteria of GM selection markers. Many countries, such as Korea, the European Union, and Japan, that have strict regulations for GM crops have serious concerns about unintended introduction of GM cultivars and food commodities using unauthorized GM crops. Transgene- and/or GM event-specific molecular markers and technologies for genomics-based detection of unauthorized GM papaya have been developed and have resulted in the robust detection of GM papayas.

Field Studios of In-situ Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbons

  • Semprini, Lewts
    • Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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    • 2004.04a
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    • pp.3-4
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    • 2004
  • Results will be presented from two field studies that evaluated the in-situ treatment of chlorinated aliphatic hydrocarbons (CAHs) using aerobic cometabolism. In the first study, a cometabolic air sparging (CAS) demonstration was conducted at McClellan Air Force Base (AFB), California, to treat chlorinated aliphatic hydrocarbons (CAHs) in groundwater using propane as the cometabolic substrate. A propane-biostimulated zone was sparged with a propane/air mixture and a control zone was sparged with air alone. Propane-utilizers were effectively stimulated in the saturated zone with repeated intermediate sparging of propane and air. Propane delivery, however, was not uniform, with propane mainly observed in down-gradient observation wells. Trichloroethene (TCE), cis-1, 2-dichloroethene (c-DCE), and dissolved oxygen (DO) concentration levels decreased in proportion with propane usage, with c-DCE decreasing more rapidly than TCE. The more rapid removal of c-DCE indicated biotransformation and not just physical removal by stripping. Propane utilization rates and rates of CAH removal slowed after three to four months of repeated propane additions, which coincided with tile depletion of nitrogen (as nitrate). Ammonia was then added to the propane/air mixture as a nitrogen source. After a six-month period between propane additions, rapid propane-utilization was observed. Nitrate was present due to groundwater flow into the treatment zone and/or by the oxidation of tile previously injected ammonia. In the propane-stimulated zone, c-DCE concentrations decreased below tile detection limit (1 $\mu$g/L), and TCE concentrations ranged from less than 5 $\mu$g/L to 30 $\mu$g/L, representing removals of 90 to 97%. In the air sparged control zone, TCE was removed at only two monitoring locations nearest the sparge-well, to concentrations of 15 $\mu$g/L and 60 $\mu$g/L. The responses indicate that stripping as well as biological treatment were responsible for the removal of contaminants in the biostimulated zone, with biostimulation enhancing removals to lower contaminant levels. As part of that study bacterial population shifts that occurred in the groundwater during CAS and air sparging control were evaluated by length heterogeneity polymerase chain reaction (LH-PCR) fragment analysis. The results showed that an organism(5) that had a fragment size of 385 base pairs (385 bp) was positively correlated with propane removal rates. The 385 bp fragment consisted of up to 83% of the total fragments in the analysis when propane removal rates peaked. A 16S rRNA clone library made from the bacteria sampled in propane sparged groundwater included clones of a TM7 division bacterium that had a 385bp LH-PCR fragment; no other bacterial species with this fragment size were detected. Both propane removal rates and the 385bp LH-PCR fragment decreased as nitrate levels in the groundwater decreased. In the second study the potential for bioaugmentation of a butane culture was evaluated in a series of field tests conducted at the Moffett Field Air Station in California. A butane-utilizing mixed culture that was effective in transforming 1, 1-dichloroethene (1, 1-DCE), 1, 1, 1-trichloroethane (1, 1, 1-TCA), and 1, 1-dichloroethane (1, 1-DCA) was added to the saturated zone at the test site. This mixture of contaminants was evaluated since they are often present as together as the result of 1, 1, 1-TCA contamination and the abiotic and biotic transformation of 1, 1, 1-TCA to 1, 1-DCE and 1, 1-DCA. Model simulations were performed prior to the initiation of the field study. The simulations were performed with a transport code that included processes for in-situ cometabolism, including microbial growth and decay, substrate and oxygen utilization, and the cometabolism of dual contaminants (1, 1-DCE and 1, 1, 1-TCA). Based on the results of detailed kinetic studies with the culture, cometabolic transformation kinetics were incorporated that butane mixed-inhibition on 1, 1-DCE and 1, 1, 1-TCA transformation, and competitive inhibition of 1, 1-DCE and 1, 1, 1-TCA on butane utilization. A transformation capacity term was also included in the model formation that results in cell loss due to contaminant transformation. Parameters for the model simulations were determined independently in kinetic studies with the butane-utilizing culture and through batch microcosm tests with groundwater and aquifer solids from the field test zone with the butane-utilizing culture added. In microcosm tests, the model simulated well the repetitive utilization of butane and cometabolism of 1.1, 1-TCA and 1, 1-DCE, as well as the transformation of 1, 1-DCE as it was repeatedly transformed at increased aqueous concentrations. Model simulations were then performed under the transport conditions of the field test to explore the effects of the bioaugmentation dose and the response of the system to tile biostimulation with alternating pulses of dissolved butane and oxygen in the presence of 1, 1-DCE (50 $\mu$g/L) and 1, 1, 1-TCA (250 $\mu$g/L). A uniform aquifer bioaugmentation dose of 0.5 mg/L of cells resulted in complete utilization of the butane 2-meters downgradient of the injection well within 200-hrs of bioaugmentation and butane addition. 1, 1-DCE was much more rapidly transformed than 1, 1, 1-TCA, and efficient 1, 1, 1-TCA removal occurred only after 1, 1-DCE and butane were decreased in concentration. The simulations demonstrated the strong inhibition of both 1, 1-DCE and butane on 1, 1, 1-TCA transformation, and the more rapid 1, 1-DCE transformation kinetics. Results of tile field demonstration indicated that bioaugmentation was successfully implemented; however it was difficult to maintain effective treatment for long periods of time (50 days or more). The demonstration showed that the bioaugmented experimental leg effectively transformed 1, 1-DCE and 1, 1-DCA, and was somewhat effective in transforming 1, 1, 1-TCA. The indigenous experimental leg treated in the same way as the bioaugmented leg was much less effective in treating the contaminant mixture. The best operating performance was achieved in the bioaugmented leg with about over 90%, 80%, 60 % removal for 1, 1-DCE, 1, 1-DCA, and 1, 1, 1-TCA, respectively. Molecular methods were used to track and enumerate the bioaugmented culture in the test zone. Real Time PCR analysis was used to on enumerate the bioaugmented culture. The results show higher numbers of the bioaugmented microorganisms were present in the treatment zone groundwater when the contaminants were being effective transformed. A decrease in these numbers was associated with a reduction in treatment performance. The results of the field tests indicated that although bioaugmentation can be successfully implemented, competition for the growth substrate (butane) by the indigenous microorganisms likely lead to the decrease in long-term performance.

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Analysis of Isolated Proteinuria on School Urinary Mass Screening Test in Busan and Kyungsangnam-do Province (학교 신체 검사에서 발견된 단독 단백뇨의 분석)

  • Oh Dong-Hwan;Kim Jung-Soo;Park Ji-Kyoung;Chung Woo-Yeong
    • Childhood Kidney Diseases
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    • v.7 no.2
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    • pp.142-149
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    • 2003
  • Purpose : The urinary mass screening program for the detection of urinary abnormalities in school aged population has been performed in Seoul since 1981. Nation-wide urinary mass screening program was also performed since 1998. The aim of this study was to analyze the cause and nature of isolated proteinuria detected by chance on the urinary mass screening test in Busan and Kyungsangnam-do Province Methods : The medical records of 44 cases of isolated proteinuria detected by chance on the urinary mass screening test in Busan and Kyungsangnam-do Province, and evaluated for urinary abnormalities at the pediatrics outpatients renal clinics of Busan Paik Hospital from April 2002 to August 2003 were reviewed prospectively. Results : The cause and incidence of isolated proteinuria were as follows; transient proteinuria 4 cases(9.1%), orthostatic proteinuria 36 cases(81.8%) and persistent proteinuria 4 cases (9.1%). The total protein amount of the 24 hour urine were $121.0{\pm}136.4\;mg$ in transient proteinuria, $179.1{\pm}130.0\;mg$ in orthostatic proteinuria and $1532.8{\pm}982.5\;mg$ in persistent proteinuria. In the orthostatic proteinuria group, the total protein amount of the 24 hour urine was in the range of 40-616 mg. Spot urine protein/creatinine ratio(PCR) were $0.10{\pm}0.01$ in transient proteinuria, $0.61{\pm}0.61$ in orthostatic proteinuria and $4.35{\pm}4.04$ in persistent proteinuria. In the orthostatic proteinuria group, spot me PCR was in the range of 0.09-2.32. Renal biopsy was peformed in 4 children of the persisitent proteinuria group. They showed minimal change in 1 case, membranoproliferatiye glomerulonephritis in 2 cases and secondary renal amyloidosis in 1 case. Conclusion : The majority of isolated proteinuria which was detected by chance on school urinary mass screening were transient or orthostatic proteinuria. Even though the incidence of persistent proteinuria was much lower, it is necessary to take care of these children regularly and continuously, because persistent proteinuria itself is a useful marker of the progressive renal problems.

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Comparative Uptake of Tc-99m Sestamibi and Tc-99m Tetrofosmin in Cancer Cells and Tissue Expressing P-Glycoprotein or Multidrug Resistance Associated Protein (P-Glycoprotein과 Multidrug Resistance Associated Protein을 발현하는 암세포와 종양에서 Tc-99m Sestamibi와 Tc-99m Tetrofosmin의 섭취율 비교)

  • Cho, Jung-Ah;Lee, Jae-Tae;Yoo, Jung-Ah;Seo, Ji-Hyoung;Bae, Jin-Ho;Jeong, Shin-Young;Ahn, Byeong-Cheol;Sohn, Sang-Gyun;Ha, Jeoung-Hee;Lee, Kyu-Bo
    • The Korean Journal of Nuclear Medicine
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    • v.39 no.1
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    • pp.34-43
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    • 2005
  • Purpose: $^{99m}Tc$-sestamibi(MIBI) and $^{99m}Tc$-tetrofosmin have been used as substrates for P-glycoprotein (Pgp) and multidrug resistance associated protein (MRP), which are closely associated with multidrug resistance of the tumors. To understand different handling of radiotracers in cancer cell lines expressing Pgp and MRP, we compared cellular uptakes of $^{99m}Tc$-MIBI and $^{99m}Tc$-tetrofosmin. The effects of cyclosporin A (CsA), well-known multidrug resistant reversing agent, on the uptake of both tracers were also compared. Materials and Methods: HCT15/CL02 human colorectal cancer cells for Pgp expressing cells, and human non-small cell lung cancer A549 cells for MRP expressing cells, were used for in vitro and in vivo studies. RT-PCR, western blot analysis and immunohistochemistry were used for detection of Pgp and MRP. MDR-reversal effect with CsA was evaluated at different drug concentrations after incubation with MIBI or tetrofosmin. Radioactivities of supernatant and pellet were measured with gamma well counter. Tumoral uptake of the tracers were measured from tumor bearing nude mice treated with or without CsA. Results: RT-PCR, western blot analysis of the cells and irnrnunochemical staining revealed selective expression of Pgp and MRP for HCY15/CL02 and A549 cells, respectively. There were no significant difference in cellular uptakes of both tracers in HCT15/CL02 cells, but MIBI uptake was slightly higher than that of tetrofosmin in A549 cells. Co-incubation with CsA resulted in a increase in cellular uptakes of MIBI and tetrofosmin. Uptake of MIBI or tetrofosmin in HCT15/CL02 cells was increased by 10- and 2.4-fold, and by 7.5 and 6.3-fold in A549 cells, respectively. Percentage increase of MIBI was higher than that of tetrofosmin with CsA for both cells (p<0.05). In vivo biodistribution study showed that MIBI (114% at 10 min, 257% at 60 min, 396% at 240 min) and tetrofosmin uptake (110% at 10 min, 205% at 60 min, 410% at 240 min) were progressively increased by the time, up to 240 min with CsA. But increases in tumoral uptake were not significantly different between MIBI and tetrofosmin for both tumors. Conclusion: MIBI seems to be a better tracer than tetrofosmin for evaluating MDR reversal effect of the modulators in vitro, but these differences were not evident in vivo tumoral uptake. Both MIBI and tetrofosmin seem to be suitable tracers for imaging Pgp- and MRP-mediated drug resistance in tumors.

Detection of Copy Number Variation of the KIT Gene in the Landrace Breed using an Quantitative Oligonucleotide Ligation Assay(qOLA) (Quantitative Oligonucleotide Ligation Assay(qOLA)를 이용한 Landrace 품종의 KIT 유전자 반복수 변이 탐지)

  • Seo, B.Y.;Kim, J.H.;Nahm, D.W.;Yoo, C.K.;Lee, S.H.;Lee, J.B.;Lim, H.T.;Jung, E.J.;Cho, I.C.;Heo, K.N.;Jeon, J.T.
    • Journal of Animal Science and Technology
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    • v.49 no.5
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    • pp.559-568
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    • 2007
  • Recently, copy number variations (CNV) of genes or genomic segments have been intensively studied and various analysis methods have been developed. In this study, quantitative oligonucleotide ligation assay (qOLA) was applied to investigate CNV of KIT gene in the Landrace breed. A combined assay using qOLA and pyrosequencing, 6 genotype classes, I1/I1 or I3/i (IBe), I1/I2 or I3/IP, I1/I3, I1/IP or I2/i (IBe), I2/I2and I2/IP, were identified from 44 Landrace pigs. Genotype assignment using grouping features of measurements on a scatter plot showed 100% agreement with those using a statistical assignment by PROC FASTCLUS procedure implemented in the SAS package. Two versions (3100 and 3130) of ABI sequencers gave the same genotyping results, indicating there was no influence on qOLA by different versions of instrument, however, the means of standard deviation and coefficient of variation from the qOLA on a ABI 3130 (2.33 and 4.10) was lower than those from the qOLA on a ABI 3100 (2.67 and 4.81). Effect of proteinase K treatment on the PCR product followed by qOLA was very clear because noise peaks were disappeared and the observed ration fit better to the reference ratio corresponding to each genotype.

Effect of the Solvent Fractions of Zingiber officinale Roscoe against Thrombintreated Tumor Invasion in Human Oral Squamous Carcinoma YD-10B Cells (YD-10B 인간구강암세포주에서 생강 유기용매 분획물의 항산화, 트롬빈억제 및 thrombin에 의해 처리된 암 침윤 및 전이 억제 효과)

  • Kim, Eun-Jung;Kim, Jun-Ho
    • Journal of Life Science
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    • v.26 no.11
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    • pp.1289-1297
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    • 2016
  • Oral squamous cell carcinoma (OSCC) is a common malignant tumor in the oral cavity, comprising up to 90% of oral cancer. Oral cancer is characterized by a marked tendency of local invasiveness and is good for early detection and treatment; therefore, it is recognized as a good model for cancer prevention. The present study investigated the antioxidant, thrombin inhibitory, and anti-invasive activities of the solvent fractions of Zingiber officinale Roscoe. Samples were fractionated into hexane, chloroform, ethyl acetate, butanol, and water fractions, and each of these was assayed individually. The water fraction showed the highest extraction yield at 9.79%(w/w). Anti-oxidative activity was analyzed by DPPH assay. Thrombin inhibitory activity was used to analyze thrombin inhibitor assay. Cell viability was detected by the MTS assay. The activity and mRNA expression of MMP-2 and MMP-9 in human oral squamous carcinoma YD-10B cells were examined by zymography and RT-PCR. The antioxidative activities of hexane and water fractions were 92.38% and 92.96%, respectively. In the thrombin inhibitory activity test, water fraction was the highest, with a value of 65.86%. MMP-2/-9 activation was increased in phorbol 12-myristate 13-acetate (PMA)-induced YD-10B cells. MMP-9 activation was increased in thrombin-treated YD-10B cells. In PMA- or thrombin-treated YD-10B cells, the increased mRNA expression and protein activation of MMP-2/-9 were significantly inhibited in the hexane fraction. Therefore, the hexane fraction obtained from a Zingiber officinale Roscoe water extract is a promising therapeutic anti-invasive agent in oral cancer.

Current status of Brassica A genome analysis (Brassica A genome의 최근 연구 동향)

  • Choi, Su-Ryun;Kwon, Soo-Jin
    • Journal of Plant Biotechnology
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    • v.39 no.1
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    • pp.33-48
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    • 2012
  • As a scientific curiosity to understand the structure and the function of crops and experimental efforts to apply it to plant breeding, genetic maps have been constructed in various crops. Especially, in the case of Brassica crop, genetic mapping has been accelerated since genetic information of model plant $Arabidopsis$ was available. As a result, the whole $B.$ $rapa$ genome (A genome) sequencing has recently been done. The genome sequences offer opportunities to develop molecular markers for genetic analysis in $Brassica$ crops. RFLP markers are widely used as the basis for genetic map construction, but detection system is inefficiency. The technical efficiency and analysis speed of the PCR-based markers become more preferable for many form of $Brassica$ genome study. The massive sequence informative markers such as SSR, SNP and InDels are also available to increase the density of markers for high-resolution genetic analysis. The high density maps are invaluable resources for QTLs analysis, marker assisted selection (MAS), map-based cloning and comparative analysis within $Brassica$ as well as related crop species. Additionally, the advents of new technology, next-generation technique, have served as a momentum for molecular breeding. Here we summarize genetic and genomic resources and suggest their applications for the molecular breeding in $Brassica$ crop.

Microbial and Pathogenic Contamination of Ready-to-eat Fresh Vegetables in Korea (한국에 유통중인 신선편이 채소류의 미생물 품질 및 병원성 세균의 오염도 조사)

  • Bae, Young-Min;Hong, Yu-Jin;Kang, Dong-Hyun;Heu, Sung-Gi;Lee, Sun-Young
    • Korean Journal of Food Science and Technology
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    • v.43 no.2
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    • pp.161-168
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    • 2011
  • The purpose of this study was to evaluate microbiological contamination of fresh vegetables in Korea. Twenty types of vegetables were tested for total aerobic bacteria, coliforms, Escherichia coli, yeast and mold, and pathogenic bacteria such as Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella, E. coli O157:H7, Cronobacter sakazakii, Shigella, and Campylobacter. Levels of total aerobic bacteria and coliform on 20 vegetables were between 3.74 and 8.04 log CFU/g, and 0.16 and 5.02 log CFU/g, respectively. The highest contamination levels of total aerobic bacteria were observed on water dropwort, sprouts, mungbean sprout, and ballflower root. There was no significant difference (p>0.05) in microbial contamination levels of total aerobic count, coliform, E. coli, yeast and mold between organic and nonorganic vegetables. When isolation methods using selective agars were applied, L. monocytogenes, B. cereus, Salmonella and Campylobacter were isolated from some fresh vegetable samples. Results of API kit tests showed that L. monocytogenes was identified on Chinese cabbage, cucumber, soybean sprouts, and iceberg lettuce while Salmonella was identified on Korean leek. Furthermore, Campylobacter jejuni was also identified in more than 50 of the 100 samples. However, when positive samples from API kit were tested for real-time PCR or 16S rRNA sequencing method, only B. cereus from perilla leaf, carrot, water dropwort, and sprouts showed positive results. These results indicate that selective agar and API kit detection methods might result in false positive results for some pathogens. Therefore, studies need to improve isolation or confirmation methods for such pathogens.