• Title/Summary/Keyword: Molecular Detection

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A Monoclonal Antibody That Specifically Binds Chitosan In Vitro and In Situ on Fungal Cell Walls

  • Schubert, Max;Agdour, Siham;Fischer, Rainer;Olbrich, Yvonne;Schinkel, Helga;Schillberg, Stefan
    • Journal of Microbiology and Biotechnology
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    • v.20 no.8
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    • pp.1179-1184
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    • 2010
  • We report the generation of the first monoclonal antibody that specifically binds to the polysaccharide chitosan. Mice were immunized with a mixture of chitosans, and hybridoma clones were screened for specific binders, resulting in the isolation of a single clone secreting a chitosan-specific IgM, mAbG7. In ELISAs, the antibody could bind to chitosans of varying composition, but demonstrated the highest affinity for chitosans with lower degrees of acetylation (DA) and very poor binding to chitin. We tested the ability of the antibody to bind to chitosan in situ, using preparations of fungal cell walls. Immunofluorescence microscopy confirmed that the antibody bound strongly to the cell walls of fungi with high levels of chitosan, whereas poor staining was observed in those species with cell walls of predominantly chitin or cellulose. The potential use of this antibody for the detection of fungal contamination and the protection of plants against fungal pathogens is discussed.

Development of a Rapid Molecular Detection Marker for Colletotrichum species with AFLP

  • Eom, Seung-Hee;Kim, Kwon-Jong;Jung, Hee-Sun;Lee, Sang-Pyo;Lee, Youn-Su
    • Mycobiology
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    • v.32 no.3
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    • pp.123-127
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    • 2004
  • Sweet persimmons have been increasingly cultivated in the southern part of Korea. However, anthracnose disease caused by Colletotrichum species is one of the major hindrances in cultivation and productions. In this study, we used polymerase chain reaction(PCR) to detect Colletotrichum species with the AFLP(amplified fragment length polymorphism) method. In AFLP, we used E3(5'-GACTGCGTACCAATTCTA-3') and M1(5'-GATGAGTCCTGAGTAACAG-3') primer combination and, as a result, 262 bp segment was observed in Colletotrichum species only. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless sweet persimmon plants. Based on sequence data for specific segments, Co.B1(5'-GAGAGAGTAGAATTGCGCTG-3') and Co.B2(5'-CTACCATTCTTCTA GGTGGG-3') were designed to detect Colletotrichum species. The 220 bp segment was observed in Colletotrichum species only, but not in other fungal and bacterial isolates.

Research Trend of Cronobacter Species Detection Methods: A Review (Cronobacter Species의 검출에 관한 연구동향: 총설)

  • Kwon, Heejun;Kim, Myunghee
    • The Korean Journal of Food And Nutrition
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    • v.28 no.4
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    • pp.728-736
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    • 2015
  • Cronobacter species (Cronobacter spp.), previously known as Enterobacter sakazakii, are gram negative food borne pathogenic bacteria. They pose a very high risk of infection to neonates and immuno-compromised individuals and can affect the human central nervous system. Consequently, survivors often suffer from severe neurological impairment including hydrocephalus, quadriplegia, and developmental delays. Cronobacter spp. were not only isolated from plant food and products such as cereals, fruits, vegetables, legume products, herbs, and spices but also from animal source foods such as milk, meat, fish, and products made from these foods. Therefore, rapid detection of Cronobacter spp. is essential for food safety. Many detection methods have been developed since the Cronobacter spp. were first reported. However, the development of more rapid, sensitive, and easy-to-use detection methods for the Cronobacter spp. is required. In this review, our aim was to study and compare the available detection methods for Cronobacter spp., including culture-based, molecular biology-based, and immunology-based methods. This study will contribute to the development of new and rapid detection method for Cronobacter spp.

Detection of Astrovirus Infection from Hospitalized Young Children Feces by Reverse Transcription-Polymerase Chain Reaction (중증 위장관염 소아의 대변검체에서 역전사-중합효소 연쇄반응을 이용한 Astrovirus 검출)

  • Park, Hae-Kyung;Woo, So-Youn;Seoh, Ju-Young;Chong, Young-Hae;Seo, Jeong-Wan
    • The Journal of the Korean Society for Microbiology
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    • v.34 no.5
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    • pp.453-459
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    • 1999
  • Astrovirus is frequently associated with diarrhea in children. It can not be readily isolated by cell culture, and an electronmicroscope is usually used for detection of this agent. Recently in 1995 a combined method of reverse transcription-polymerase chain reaction (RT-PCR) was designed for easier detection of astrovirus, which is based on the conserved sequence in 3'-end of genomes of the 7 known serotypes of human astrovirus. As of yet there has not been any report of astrovirus data in Korea using the RT-PCR methods. The purpose of this study was to detect astrovirus incidence, severity of symptoms, seasonal variation and co infection rate with rotavirus in Korean children inpatients with diarrhea. Fecal specimens from 61 young children hospitalized with gasteroenteritis Korea from Jan. 1996 through Mar. 1997. They were examined for astroviurs infection by RT-PCR method. Results are as follows:1. Astrovirus was detected at 9.8% (6/61) from fecal specimens of children with severe diarrhea by EIA using monoclonal antibody coated plates. 2. Astorvirus was detected at 29.5% (18/61) from fecal specimens of children with severe diarrhea by RT-PCR. 3. The age of the 18 children affected by astrovirus ranged from 2 monthes to 7 years with mean of 3.0 years. 4. Mean hospital stay of the 18 children was 6.1 days. 5. Five (27.8%) astrovirus RT-PCR positive strains were confirmed in November and in December, respectively out of 18 specimens in total. 6. Astrovirus coinfection with rotavirus type G1 was confirmed in 15/16 specimens (93.8%), and with type G2 was in 1/16 specimens (6.3%).

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Pulsed Field Gel Electrophoresis Profile of Erythromycin-Clindamycin Resistant Streptococcus pyogenes Isolated in Korea (국내분리 Erythromycin-Clindamycin 내성 Streptococcus pyogenes에 대한 Pulsed Field Gel Electrophoresis 양상 분석)

  • Lee, Young-Hee;Hwang, Kyu-Jam;Lee, Kwang-Jun;Park, Kang-Soo;Bae, Song-Mee;Sung, Hwa-Young;Kim, Ki-Sang;Lee, Chong-Sam
    • The Journal of the Korean Society for Microbiology
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    • v.35 no.2
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    • pp.171-180
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    • 2000
  • Ninety two strains of Streptococcus pyogenes were isolated from patients with pharyngitis, scarlet fever, skin infection, and invasive streptococcal infections in Seoul, Korea from January to December, 1998. All isolates were epidemiologically characterized by T protein serotype, and serum opacity factor (OF) detection to phenotypes. To analyze the genetic relationship, fifty two isolates including 32 erythromycin-clindamycin (Em-Cm) resistant strains, 20 antimicrobial susceptible strains were attempted to the pulsed-field gel electrophoresis (PFGE). T protein serotype showed 16 kinds in distribution including T12 and T4. Among the total isolates, 40 strains (43.5%) belonged to the T12 serotype and twenty strains (21.7%) to T4 serotype. On the other hand, when infection aspect of S. pyogenes isolates were analysed by T serotype distribution, T12 type was predominant for pharyngitidis which contributed to 21 strains (53%) and for skin infection isolates which contributed to 11 strains (28%), respectively. In case of T4 type, it was the most predominant pharyngitidis isolates which contributed to 8 strains (40%). In T serotype distribution of Em-Cm resistant strains, 27 strains (84%) of the thirty two showed T12 serotype. In minimum inhibitory concentration (MIC) values of Em-Cm resistance isolates, thirty two isolates showed resistant to erythromycin 27 strains (84%), had high MIC of >$128\;{\mu}g/ml$. And also to clindamycin, twenty two strains (69%) had high MIC of >$128\;{\mu}g/ml$. When OF detection of Em-Cm resistance of S. pyogenes isolates were analyzed by T serotype distribution, T12 serotype isolates revealed that all of the isolates except one strain were OF negative. In PFGE profile analysis to Em-Cm resistance isolates, of the twenty seven, Em-Cm resistance of T12 serotype isolates, 26 strains showed identical PFGE profile and all of these isolates revealed that OF negative. Eighty four percent of Em-Cm resistance S. pyogenes isolates had identical phenotype and PFGE profile. These results strongly suggested that the Em-Cm resistant S. pyogenes isolates from Seoul area showed close genetic correlation and PFGE could be available tool for molecular epidemiology.

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Chemical properties of cores in different environments; the Orion A, B and λ Orionis clouds

  • Yi, Hee-Weon;Lee, Jeong-Eun;Tie, Liu;Kim, Kee-Tae
    • The Bulletin of The Korean Astronomical Society
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    • v.42 no.2
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    • pp.80.1-80.1
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    • 2017
  • We present preliminary results of KVN single dish telescope observations of 80 dense cores in the Orion molecular cloud complex which contains the Orion A, B, and ${\lambda}$ Orionis cloud. We investigate the behavior of the different molecular tracers and look for chemical variations of cores in the three clouds in order to systematically investigate the effects of stellar feedback. The most commonly detected molecular lines (with the detection rates higher than 50%) are N2H+, HCO+, H13CO+, C2H, HCN, and H2CO. The detection rates of dense gas tracers, N2H+, HCO+, H13CO+, and C2H show the lowest values in the ${\lambda}$ Orionis cloud. We find difference between molecular D/H ratios and N2H+/H13CO+ abundance ratios towards different clouds, and between protostellar cores and starless cores. Eight starless cores in the Orion A and B clouds exhibit high deuterium fractionations, larger than 0.10, while in the ${\lambda}$ Orionis cloud, no cores reveal the high ratio. These chemical properties could support that cores in the ${\lambda}$ Orionis cloud are affected by the photo-dissociation and external heating from the nearby H II region, which is a hint of negative stellar feedback on core formation. The striking difference between the [N2H+]/[H13CO+] ratios leads us to suggest that there are significant evolutionary differences between the Orion A/B and ${\lambda}$ Orionis clouds. In order to examine whether starless cores can be candidates of pre-stellar cores, we compared the core masses estimated from the 850 um emission to their Virial masses calculated from the N2H+ line data and find that most of them are not gravitationally bound in the three clouds.

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Comparison of EMA,CEA, CD10 and Bcl-2 Biomarkers by Immunohistochemistry in Squamous Cell Carcinoma and Basal Cell Carcinoma of the Skin

  • Ramezani, Mazaher;Mohamadzaheri, Elham;Khazaei, Sedigheh;Najafi, Farid;Vaisi-Raygani, Asad;Rahbar, Mahtab;Sadeghi, Masoud
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.3
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    • pp.1379-1383
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    • 2016
  • Background: Basal and squamous cell carcinoma (BCC and SCC) are the most common skin cancers worldwide and distinction between the two may sometimes be very difficult in routine histopathology. The present study was aimed to evaluate a reliable diagnostic method for these cancers based on immunohistochemistry (IHC). Materials and Methods: IHC was used with antibodies to Bcl-2, CD10, CEA, and EMA biomarkers, which despite non-specificity are easily available for detection of various types of tumors in pathology sections and can be used as a panel for differentiation. In this descriptive and analytic study, paraffin-embedded blocks of 29 SCC patients and 29 BCC patients were collected and sectioned for IHC staining. The results were analyzed by the STATA (version 8) statistical package using the Chi-square test. Results: BCC patients were 100%, 75.8%, 0% and 0% positive for Bcl-2, CD10, CEA and EMA markers, respectively, and for SCC patients were 3.5%, 0%, 34.5% and 82.7% positive, respectively. Using simultaneously Bcl-2 and CD10 as positive markers, detection of BCC with 88% accuracy and 100% specificity was possible, while application of CEA and EMA positivity could detect SCC with 67% accuracy and 100% specificity. Conclusions: SCC and BCC have different immunostaining profiles; therefore, Bcl-2, CD10, CEA and EMA markers can be helpful to distinguish between them.

Detection of Serum IgE Specific to Mite Allergens by Immuno-PCR

  • Lee, Kyung-Woo;Hur, Byung-Ung;Chua, Kaw-Yan;Kuo, I-Chun;Song, Suk-Yoon;Cha, Sang-Hoon
    • IMMUNE NETWORK
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    • v.8 no.3
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    • pp.82-89
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    • 2008
  • Background: Although a skin test is the primary option for detecting allergen-specific IgE in clinics, the serum IgE immunoassay is also important because it allows for the diagnosis of allergy without any accompanying adverse effect on the patient. However, the low detection limit of IgE levels by immunoassay may restrict the use of the method in some occasions, and improving its sensitivity would thus have a significant implication in allergy-immunology clinics. Methods: In this study, we attempted to detect specific serum IgE by using immuno-polymerase chain reaction (IPCR) which combines the antigen-antibody specificity of enzyme-linked immunosorbent assays (ELISAs) with the amplification power of PCR. Results: Our results demonstrated that Blo t5-specific serum IgE can be detected by IPCR with a 100-fold higher sensitivity than ELISA, and cross-reactivity of serum IgE to other mite allergens is able to be analyzed by using only $0.3{\mu}l$ of serum sample. Use of real-time IPCR seemed to permit more convenient determination of specific serum IgE as well. Conclusion: We believe that IPCR can serve as a valuable tool in determining specific serum IgE, especially when the amount of serum sample is limited.

Detection of Abnormally High Amygdalin Content in Food by an Enzyme Immunoassay

  • Cho, A-Yeon;Yi, Kye Sook;Rhim, Jung-Hyo;Kim, Kyu-Il;Park, Jae-Young;Keum, Eun-Hee;Chung, Junho;Oh, Sangsuk
    • Molecules and Cells
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    • v.21 no.2
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    • pp.308-313
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    • 2006
  • Amygdalin is a cyanogenic glycoside compound which is commonly found in the pits of many fruits and raw nuts. Although amygdalin itself is not toxic, it can release cyanide (CN) after hydrolysis when the pits and nuts are crushed, moistened and incubated, possibly within the gastrointestinal tract. CN reversibly inhibits cellular oxidizing enzymes and cyanide poisoning generates a range of clinical symptoms. As some pits and nuts may contain unusually high levels of amygdalin such that there is a sufficient amount to induce critical CN poisoning in humans, the detection of abnormal content of amygdalin in those pits and nuts can be a life-saving measure. Although there are various methods to detect amygdalin in food extracts, an enzyme immunoassay has not been developed for this purpose. In this study we immunized New Zealand White rabbits with an amygdalin-KLH (keyhole limpet hemocyanin) conjugate and succeeded in raising anti-sera reactive to amygdalin, proving that amygdalin can behave as a hapten in rabbits. Using this polyclonal antibody, we developed a competition enzyme immunoassay for determination of amygdalin concentration in aqueous solutions. This technique was able to effectively detect abnormally high amygdalin content in various seeds and nuts. In conclusion, we proved that enzyme immunoassay can be used to determine the amount of amygdalin in food extracts, which will allow automated analysis with high throughput.

Detection of plcR-papR Genes by PCR in Identifying Enterotoxin Genes-Harboring Bacillus cereus Strains (장독소 유전자 함유 Bacillus cereus 확인을 위한 독소 전사 조절 유전자 plcR-papR의 PCR 검출법)

  • Yun, Suk-Hyun;Kim, Yong-Sang;So, Soon-Ku;Jeong, Do-Yeon;Hahn, Kum-Su;Uhm, Tai-Boong
    • Korean Journal of Microbiology
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    • v.45 no.4
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    • pp.425-429
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    • 2009
  • Identification of virulent Bacillus cereus strains was examined by PCR using primers specific for the detection of plcR-papR, which encode regulatory proteins controlling the transcription of virulence factors in B. cereus. Total 96 strains of B. cereus that carried at least one of diarrheal toxin genes including hblACD, nheABC, and cytK showed all positive PCR products, while other 48 Bacillus strains that lacked the toxin genes were plcRpapR-negative. This PCR method targeting the plcR-papR genes appears to be simple and effective in identifying the enterotoxin genes-harboring B. cereus strains.