• Title/Summary/Keyword: Strain Amplification

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Sequence and Phylogenetic Analysis of Respiratory Syncytial Virus Isolated from Korea (국내에서 유행한 Respiratory Syncytial 바이러스의 염기서열 및 계통분석)

  • Kwon, Soon-Young;Choi, Young-Ju;Kim, So-Youn;Song, Ki-Joon;Lee, Yong-Ju;Choi, Jong-Ouck;Seong, In-Wha
    • The Journal of Korean Society of Virology
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    • v.26 no.1
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    • pp.9-22
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    • 1996
  • Respiratory Syncytial virus (RSV) is an important cause of acute lower respiratory tract infections in human, with infants and young children being particularly susceptible. In the temperate zones, sharp annual outbreaks of RSV occur during the colder months, in both the northern and the southern hemisphere. RSV is unusual in that it can repeatedly reinfect individuals throughout life and infect babies in the presence of maternal antibody. RSV isolates can be divided into two subgroups, A and B, on the basis of their reactions with monoclonal antibodies, and the two subgroups are also distinct at the nucleotide sequence level. The specific diagnosis of RSV infection was best made by isolation of virus in tissue culture, identification of viral antigen, or by specific serologic procedures. Recently, rapid detection of RSV and analysis of RSV strain variation became possible by development of methods of reverse transcription and polymerase chain reaction amplification. In this study, to determine the genetic diversity of RSV found in Korea, 173 bp and 164 bp spanning selected regions of the RSV F and SH genes were enzymatically amplified and sequenced, respectively. Eight for F gene and three for SH gene were detected in 66 nasopharyngeal swap samples tested. Two major antigenic subgroups, A and B were confirmed from Korean samples (seven for subgroup A and one for subgroup B). At the nucleotide level of the F gene region, Korean subgroup A strains showed 95-99% homologies compared to the prototype A2 strain of subgroup A and 93-100% homologies among Korean subgroup A themselves. For the SH gene region, Korean subgroup A strain showed 97.5% homology compared to the prototype A2 strain of subgroup A, and Korean subgroup B strain showed 97% homology compared to the prototype 18537 strain of subgroup B. Most of base changes were transition and occured in codon position 3, which resulted in amino acid conservation. Using the maximum parsimony method, phylogenetic analysis indicated that Korean RSV strains formed a group with other RSV strains isolated from the United States, Canada, the Great Britain and Australia.

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Amplified fragment length polymorphism fingerprinting analysis of Staphylococcus aureus isolated from bovine mastitis milk (소 유방염 유래 Staphylococcus aureus의 AFLP 지문분석)

  • Kim, Yeon-soo;Kim, Sang-kyun;Hwang, Eui-kyung
    • Korean Journal of Veterinary Research
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    • v.41 no.2
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    • pp.157-165
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    • 2001
  • Amplified fragment length polymorphism(AFLP) technique is based on the polymorphism detection through selective PCR amplification of restriction fragments from digested genomic DNA and thus includes the procedures of the total DNA digestion by endonucleases, ligation of adapters to the ends of the fragments, and following the selective amplification of the restricted DNA fragments. This study were aimed to : (1) determine the genetic variability of S aureus strains, (2) estimate genetic diversity within and among these strains, (3) compare phylogenetic relationships among these strains as genetic markers using AFLP techniques. Genomic DNA was digested with a particular combination of three restriction enzymes with specific recognition sites and the DNA fragments were ligated to restriction specific adapters and amplified using the selective primer combinations. In the S aureus strain, the number of scorable AFLP bands detected per each primer combination varied from 29 to 102, with an average of 61.59 using 27 primer combinations. A total of 1,663 markers were generated, 904 bands of which were polymorphic, showing a 33.48% level of polymorphism with these primer combinations. Among the primer combinations, E02/T02, E02/T03, E04/H02, E02/T01 and E04/H03 primer combinations showed a high level of polymorphism with 0.78, 0.76, 0.74, 0.71 and 0.70, respectively. But T03/H01, E01/T02 and E01/T03 primer combinations showed a low level of polymorphism with 0.38, 0.37 and 0.15, respectively, Therefore, the former primer combinations will be the most effective for AFLP analysis of S aureus. In SA1 sub-types the level of polymorphism of S aureus KCTC 1927 was similar to that of S aureus CU 01(0.825) and higher than those of other strains such as S aureus CU 02 (0.715), S aureus KCTC 2199(0.625), S aureus KCTC 1916(0.607) and S aureus KCTC 1621 (0.553). In SA2 sub-types the level of polymorphism of S aureus CU 07 was similar to that of S aureus CU 08(0.935) and higher than those of both S aureus CU 04(0.883) and S aureus CU 05(0.883) and lower than those of S aureus CU 03(0.583). In SA3 subtypes the level of polymorphism of S aureus CU 11 was similar to that of S aureus CU 12(0.913) and lower than that of S aureus CU 15(0.623). The results proved that AFLP marker analysis of S aureus strain could be used to study the epidemiology of mastitis and in addition, common genotype in geographic region could be useful for the development of an effective vaccine or DNA marker for easy diagnosis of mastitis caused by S aureus infection.

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Monitoring of Bifenazate Resistant Two-spotted Spider Mite, Tetranychus urticae Using Molecular Detection Method (분자학적 진단방법을 이용한 bifenazate 저항성 점박이응애 모니터링)

  • Lee, Kyu-Ri;Shin, Yun-Ho;Cho, Sun-Ran;Koo, Hyun-Na;Choi, Jang-Jeon;Ahn, Ki-Su;Kim, Gil-Hah
    • The Korean Journal of Pesticide Science
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    • v.15 no.1
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    • pp.61-67
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    • 2011
  • In 2010, two-spotted spider mite, Tetranychus urticae was collected from the rose greenhouse and apple orchards in Cheongju (CJ), Chungju (CUJ)-1, CUJ-2, Kangjin (KJ), Yesan (YS), and Yeongju (YJ). Among them, KJ and YS strain showed high resistance to bifenazate of 964.5- and 1l30-fold, respectively. The other strains showed low resistance to bifenazate. By analyzing the mitochondrial cytochrome b (cytb) sequence, G126S point mutation was detected in KJ and YS strain. Thus, G126S point mutation in the mitochondrial cytb was available molecular detection marker for selection of bifenazate resistant T. urticae. Two molecular detection methods, quantitative sequencing (QS) and PCR amplification of specific alleles (PASA) were well detected specific G126S point mutation. Therefore, these methods can be used to monitor the resistance allele in field population of T. urticae and bifenazate resistance management strategy.

Impact Evaluation of Rubber Type, Hardness and Induced Prestress Force on the Dynamic Properties of a Damper (감쇠장치의 동적특성에 대한 고무의 종류, 경도 및 프리스트레스력의 영향 평가)

  • Im, Chae-Rim;Yang, Keun-Hyeok;Mun, Ju-Hyun;Won, Eun-Bee
    • Journal of the Korea institute for structural maintenance and inspection
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    • v.26 no.6
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    • pp.167-174
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    • 2022
  • The objective of this study is to evaluate the dynamic properties of DUS (damping-up system) composed of the materials with excellent damping capacity, and to compare with those of the conventional hangar bolt. The main parameters are the type and hardness (𝜂H), of rubber and the prestress force (value converted from the compression strain (𝜂R) in the stress-strain relationship of rubber). The dynamic properties were examined from the natural frequency (𝜔n), maximum response acceleration (Am), amplification coefficient (𝛼p), maximum relative displacement (𝚫m), and damping ratio (𝜉D). The test results showed that the Am, 𝛼p, and 𝚫m values of DUS were 46.3%, 46.6% and 62.9% lower, respectively, and the 𝜉D value was 3.89 times higher, when compared to those of the conventional hangar bolt. In particular, the 𝛼p value was 1.3 for DUS, and 2.45 for the conventional hanger bolt, which were similar to those of rigid and flexible components specified in KDS 41 17 00, respectively. Consequently, in the optimal details of DUS, the 𝜂H values of 50 and 45 were required for the NR (natural rubber) and EPDM (ethylene propylene diene monomer), and the 𝜂R value of 5% was also recommended.

Detection of Salmonella Using the Loop Mediated Isothermal Amplification and Real-time PCR (등온 증폭법과 Real-time PCR을 이용한 Salmonella 검출)

  • Ahn, Young-Chang;Cho, Min-Ho;Yoon, Il-Kyu;Jung, Duck-Hyun;Lee, Eun-Young;Kim, Jin-Ho;Jang, Won-Cheoul
    • Journal of the Korean Chemical Society
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    • v.54 no.2
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    • pp.215-221
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    • 2010
  • Salmonella is an important food-and water-borne pathogen associated with acute gastrointestinal illnesses around the world. The most common serotypes isolated from humans are Salmonella enterica serotype Typhimurium (S. Typhimurium) and S. Enteritidis. Traditional detection methods for Salmonella are based on cultures using selective media and characterization of suspicious colonies by biochemical and serological tests. These methods are generally time-consuming and not so highly sensitive. Recently, the Loop Mediated Isothermal Amplification and real-time PCR has been used as a highly sensitive, specific, and rapid test for the presence of pathogenic bacteria. In this study, a LAMP and real-time PCR was used to detect S. Typhimurium and S. Enteritidis. We selected target genes, which were the in invA and a randomly cloned sequence specific for the genus Salmonella. With LAMP and real-time PCR, random sequence was detected from Salmonella spp, invA were detected from all strain of S. Typhimurium and S. Enteritidis. This assay indicate that the specificity, sensitivity and rapid of the LAMP and real-time PCR make them potentially valuable tools for detection of S. Typhimurium and S. Enteritidis.

Selection of Insecticide Resistance Markers in Field-collected Populations of Myzus persicae (복숭아혹진딧물 야외개체군의 살충제 저항성 마커 선발)

  • Kim, Ju Il;Kwon, Min;Shim, Jae Dong;Kim, Jeom Soon;Lee, Yeong Gyu;Jee, Sam Nyu;Lee, Jeong Tae;Ryu, Jong Soo;Yoo, Dong Lim;Lee, Gye Jun
    • Korean journal of applied entomology
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    • v.53 no.2
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    • pp.149-156
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    • 2014
  • The resistance levels of the green peach aphid, Myzus persicae (Sulzer), against 10 insecticides was checked and selected the applicable insecticide resistance markers. We conducted our study in 5 cabbage cultivation regions (Pyeongchang, Hongcheon, Bongwha, Muju, and Jeju) of Korea, over 3 successive years (2009-2011). We selected a multi-resistant (MR) strain from among the 5 field-collected populations. We analyzed esterase over-expression and mutation(s) in the target sites, by using native isoelectric focusing (IEF) and quantitative sequencing (QS). We detected esterase over-expression and StoF mutation in the acetylcholinesterase 1 gene (ace1) in all of the field-collected populations, including the MR strain. We did not detect the LtoF mutation, which is a well-known knockdown resistance (kdr) mutation in the para-type sodium channel gene (para), in the MR strain; however, the value of the MR strain for bifenthrin was 3,461-fold higher than that of the susceptible strain. Our results indicate that insecticide resistance is more effectively evaluated using molecular markers than by conducting a bioassay. The molecular markers StoF in ace1 and MtoL in para can easily be applied in diagnostic methods such as QS or PCR amplification of specific alleles (PASA). These methods may be extended to management of M. persicae resistance in the field.

Molecular Characterization of a Korean Isolate of Human Norovirus, the Hu/NLV/Gunpo/2006/KO Strain (인체 노로바이러스의 한국분리주 Hu/NLV/Gunpo/2006/KO의 분자생물학적 특성)

  • Jeong, Ah-Yong;Yun, Sang-Im;Jee, Young-Mee;Kang, Yoon-Sung;Lee, Young-Min
    • Korean Journal of Microbiology
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    • v.45 no.2
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    • pp.105-111
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    • 2009
  • Norovirus (NV) with a variety of genotypes, a member of the family Caliciviridae, causes acute nonbacterial gastroenteritis in humans. We determined the nucleotide sequence of three open reading frames (ORFs) of a NV Korean strain and characterized the genetic relationship with others. The Korean strain designated Hu/NLV/Gunpo/2006/KO was isolated from the stool specimen of a 2-year-old female suffering from gastroenteritis. By performing reverse transcription and PCR amplification, three overlapping cDNAs were synthesized and used for direct sequencing. We found that like other NVs, this strain contains three ORFs: ORF1, 5,100 bp; ORF2, 1,647 bp; ORF3, 765 bp. Of 35 NVs, ORF1 had a level of genetic diversity lower than ORF2 and ORF3, of which the C-termini of the ORF2 and ORF3 showed a relatively high degree of genetic diversity. Phylogenetic analyses indicated that the Korean strain belonged to genogroup II, with Saitama U1, Gifu'96, Mc37, and Vietnam 026 being formed a single genetic cluster. The nucleotide sequence information of three ORFs of a NV Korean isolate will be useful not only for the development of a diagnostic tool and understanding of genetic relationship, but also provide important basic information for the functional analysis of their gene products.

Endo-1,4-β-xylanase B from Aspergillus cf. niger BCC14405 Isolated in Thailand: Purification, Characterization and Gene Isolation

  • Krisana, Asano;Rutchadaporng, Sriprang;Jarupan, Gobsuk;Lily, Eurwilaichitr;Sutipa, Tanapongpipat;Kanyawim, Kirtikara
    • BMB Reports
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    • v.38 no.1
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    • pp.17-23
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    • 2005
  • During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of $55^{\circ}C$. When tested using xylan from birchwood, it showed $K_m$ and $V_{max}$ values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by $CuSO_4$, EDTA, and by $FeSO_4$. The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-$\beta$-xylanase. The full-length gene encoding endo-1,4-$\beta$-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-$\^{a}$-xylanase B previously identified in A. niger.

Characterization of a Strain of Malva Vein Clearing Virus in Alcea rosea via Deep Sequencing

  • Wang, Defu;Cui, Liyan;Pei, Yanni;Ma, Zhennan;Shen, Shaofei;Long, Dandan;Li, Lingyu;Niu, Yanbing
    • The Plant Pathology Journal
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    • v.36 no.5
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    • pp.468-475
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    • 2020
  • Malva vein clearing virus (MVCV) is a member of the Potyvirus species, and has a negative impact on the aesthetic development of Alcea rosea. It was first reported in Germany in 1957, but its complete genome sequence data are still scarce. In the present work, A. rosea leaves with vein-clearing and mosaic symptoms were sampled and analyzed with small RNA deep sequencing. By denovo assembly the raw sequences of virus-derived small interfering RNAs (vsiRs) and whole genome amplification of malva vein cleaning virus SX strain (MVCV-SX) by specific primers targeting identified contig gaps, the full-length genome sequences (9,645 nucleotides) of MVCV-SX were characterized, constituting of an open reading frame that is long enough to encode 3,096 amino acids. Phylogenetic analysis showed that MVCV-SX was clustered with euphorbia ringspot virus and yam mosaic virus. Further analyses of the vsiR profiles revealed that the most abundant MVCV-vsiRs were between 21 and 22 nucleotides in length and a strong bias was found for "A" and "U" at the 5′-terminal residue. The results of polarity assessment indicated that the amount of sense strand was almost equal to that of the antisense strand in MVCV-vsiRs, and the main hot-spot region in MVCV-SX genome was found at cylindrical inclusion. In conclusion, our findings could provide new insights into the RNA silencing-mediated host defence mechanism in A. rosea infected with MVCV-SX, and offer a basis for the prevention and treatment of this virus disease.

Nucleotide Sequence of Coat Protein Gene of Kyuri Green Mottle Mosaic Virus Isolated from Zucchini

  • Lee, Su-Heon;Lee, Young-Gyu;Park, Jin-Woo;Park, Hong-Soo;Kim, Yeong-Tae;Cheon, Jeong-Uk;Lee, Key-Woon
    • The Plant Pathology Journal
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    • v.16 no.2
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    • pp.118-124
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    • 2000
  • The coat protein (CP) gene of kyuri green mottle mosaic virus zucchini strain (KGMMV-Z) isolated from zucchini (Cucurbita pepo) in Chonfu, Korea in 1999 was sequenced by the reverse transcription and polymerase chain reaction with degenerate and generate primers originated from tobamoviruses. The degenerate primers were very effective in amplification of KGMMV-Z CP region. The KGMMV-Z CP gene consisted of 486 nucleotides and had the same nucleotide length compared with those of cucurbit-infecting tobamoviruses. KGMMV-Z CP gene shared 43.8, 44.2, and 44.4% nucleotide sequence similarity with the CP gene of cucumber green mottle mosaic virus watermelon strain (CGMMZ-W), CGMMV-KW1, and CGMMV-SH, respectively, whereas three CGMMV strains among themselves showed 98.6-99.6% nucleotide similarity. The deduced amino acids of KGMMV-Z CP gene were 161 amino acid residues with the molecular weight of 17,181 daltons. The first 24 codons of KGMMV-Z CP gene corresponded to the sequences of the N-terminal amino acid of the viral capsid protein. The amino acid sequences of KGMMV-Z CP had 45.3% similarity compared with those of three CGMMV strains. However, the amino acid sequences of CGMMV strains were identical. These results showed that two cucurbit-infecting tobamovirus members, KGMMV-Z and CGMMV were genetically distantly related.

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