• Title/Summary/Keyword: Species-specific PCR

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Analysis of Vaginal Lactic Acid Producing Bacteria in Healthy Women

  • Nam, Hye-Ran;Whang, Kyung-Hee;Lee, Yeon-Hee
    • Journal of Microbiology
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    • v.45 no.6
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    • pp.515-520
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    • 2007
  • Vaginal lactic acid-producing bacteria of 80 pre-menopausal women were studied by isolation on Blood and DeMan-Rogosa-Sharpe agar, PCR with group-specific primers for Lactobacillus-denaturing gradient gel electrophoresis (DGGE), and PCR with specific primers for V3 region in 16S rRNA-temporal temperature gel electrophoresis (TTGE). Conventional isolation method on media detected only one lactobacillus (Lactobacillus brevis) while TTGE detected only Lactobacillus sp. DGGE detected seven Lactobacillus species; L. coleohominis, L. crispatus, L. iners, L. reuteri, L. rhamnosus, L. vaginalis, and Leuconostoc lactis. L. acidophilus and L. gasseri, which are prevalent in Western women, were not detected in Korean women. Furthermore, L. rhamnosus, Leuc. lactis, L. coleohominis, and Weissella cibaria, which were not previously reported in the vaginal microbiota of Korean women, were detected. The five most prevalent LABs in vaginal microbiota in Korean women were L. iners, Enterococcus faecalis, L. crispatus, Leuc. lactis, and W. cibaria.

Species-specific Detection of Erwinia pyrifoliae by PCR Assay Using Enterobacterial Repetivive Intergenic Consensus (ERIC) Primers

  • Park, Duck-Hwan;Thapa, Shree Prasad;Kim, Won-Sik;Hur, Jang-Hyun;Lim, Chun-Keun
    • The Plant Pathology Journal
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    • v.26 no.3
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    • pp.267-270
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    • 2010
  • We designed a sensitive and specific PCR-based method with enterobacterial repetitive intergenic consensus (ERIC) primer to detect Erwinia pyrifoliae, which cause shoot blight in Asian pear, from a mixed culture and infected plant materials. The primers specifically detected only E. pyrifoliae and showed no cross-reactivity with other bacterial phytopathogens.

Specific Marker Gene Analyses for DNA Polymorphism of the Blood Cell in Korea Native Brindled Cattle (칡한우 혈액에서 DNA 다양성 분석을 통한 표지 유전자 탐색)

  • Kim, Sang-Hwan;Hong, Yeon-Sik;Lee, Ho-Joun;Yoon, Jong-Taek
    • Development and Reproduction
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    • v.15 no.4
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    • pp.315-324
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    • 2011
  • This study was conducted to detect the specific expressing genes by using RAPD-PCR and RFLP method in the Korea Native Brindled Cattle, Korean Native cow and Holstein cattle. And then, the specific marker gene was investigated by the analysis of the genes for detection significance according to the expressing pattern. We found the specific expression gene by the RAPD-PCR analysis in Korea Native Brindled Cattle. It was detected the differences of the species in the colour and external section. The Korea Native Brindled Cattle were vary low compare to the Korean Native cow and Holstein cattle by analysis result of polymorphism and distribution. And there were a found the specific marker gene by sequencing in the R9B gene fragment of Korea Native Brindled Cattle. And the sequencing result of the R9B was different between Korean Native cow and Holstein cattle. Thus, this gene can be apply as the specific marker gene in the Korea Native Brindled Cattle.

Evaluation of Various PCR Assays for Detection of Emetic-Toxin-Producing Bacillus cereus

  • Kim, Jung-Beom;Kim, Jae-Myung;Park, Yong-Bae;Han, Jeong-A;Lee, Soon-Ho;Kwak, Hyo-Sun;Hwang, In-Gyun;Yoon, Mi-Hye;Lee, Jong-Bok;Oh, Deog-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.20 no.7
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    • pp.1107-1113
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    • 2010
  • Because conventional methods for detecting emetic-toxin-producing B. cereus are laborious and costly, various PCR assays, which are easy and cheap, have recently been reported. Therefore, this study estimated and compared the ability of various PCR assays to detect emetic-toxin-producing B. cereus strains isolated in Korea. The PCR assays were performed on 160 B. cereus strains, including 40 emetic-toxin-producing strains. Although the species-specific PCR assays were all shown to be highly specific, the sensitivities varied greatly. The accuracies of the primers were 97.5% (CER), 95.6% (EM1), 96.3% (RE234), 89.4% (CES), and 83.1% (Ces3R/CESR2). Moreover, the CER primer had a higher sensitivity (100%) than all the other primers tested, and a specificity of 96.7%. Thus, the CER primer was shown to be the most effective for screening the emetic-toxin-producing B. cereus strains tested in this study. However, the ability of these PCR assays to identify emetic-toxin-producing B. cereus should also be confirmed using other methods.

Molecular Identification of Arbuscular Mycorrhizal Fungal Spores Collected in Korea

  • Lee, Jai-Koo;Park, Sang-Hyeon;Eom, Ahn-Heum
    • Mycobiology
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    • v.34 no.1
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    • pp.7-13
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    • 2006
  • Arbuscular mycorrhizas (AM) have mutualistic symbiosis with plants and thus efforts have been placed on application of these symbiotic relationships to agricultural and environmental fields. In this study, AM fungi were collected from 25 sites growing with 16 host plant species in Korea and cultured with Sorghum bicolor in greenhouse condition. AM fungal spores were extracted and identified using both morphological and molecular methods. Using morphological characters, total 15 morpho-speices were identified. DNA was extracted from single spore of AM fungi and a partial region on 18S rDNA was amplified using nested PCR with AM fungal specific primers AML1/AML2. A total of 36 18S rDNA sequences were analyzed for phylogenetic analysis and 15 groups of AM fungi were identified using both morphological and molecular data of spores. Among the species, 4 species, Archaeospora leptoticha, Scutellospora castanea, S. cerradensis, S. weresubiae were described for the first time in Korea and two species in Glomus and a species in Gigaspora were not identified. Morphological and molecular identification of AM fungal spores in this study would help identify AM fungal community colonizing roots.

Identification of Different Species and Dultivars of Brassica by SDS-PAGE, Isozyme and Molecular Marker

  • Mukhlesur Rahman Md.;Hirata Yutaka
    • Journal of Plant Biotechnology
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    • v.7 no.1
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    • pp.27-35
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    • 2005
  • Eighty-five different cultivars of Brassica rapa, B. juncea, B. nap us, B. carinata, B. oleracea and hexaploid Brassica collected from Bangladesh, Japan, China and Denmark were analyzed by SDS-PAGE for seed and leaf protein variations, using esterase, acid phosphatase and peroxidase isozyme analysis. Ten polymorphic bands were identified from seed protein however no identifiable polymorphic band was found in the leaf protein. Polymorphic markers clearly distinguished the different Brassica species as well as yellow sarson (YS) and brown seeded (BS) cultivars of B. rapa. The $F_1$ cross between YS and brown seeded cultivars showed the existance of all poly-morphic bands of the respective parents. The Bangla-deshi and Japanese cultivars of B. rapa differed in the amount of seed protein. In the case of isozyme analysis, esterase showed the highest number of polymorphic bands (13) followed by acid phosphatase (9) and peroxidase (5). These polymorphic markers were very effec-tive for classification of all the species studied in this experiment. In parentage tests using isozymes, the hybridity of intra-and-interspecific crosses of almost all the seedlings could be identified from their respective cross combinations. Esterase polymorphism showed a clear differentiation between YS and BS types of B. rapa. In addition, two esterase polymorphic markers were iden ified to differentiate some cultivars of B. juncea. Segregation patterns in these two esterase bands showed a simple Mendelian monohybrid ratio of 3:1 in $F_2$, 1:1 in test cross and 1:0 in back cross progenies. No polymorphic band was identified to distinguish different cultivars of the same species by acid phosphatase or peroxidase. Polymerase Chain Reaction (PCR) was carried out with seed coat color specific marker of B. juncea. The yellow seeded cultivars produced a strong band at 0.5 kb and weak band 1.2 kb. In the addition of these two specific bands, Japanese yellow-seeded cultivars expressed two more weak bands at 1.0 kb and 1.1 kb. Where the brown seeded cultivars generated a single strong band at 1.1 kb. In segregating population, the yellow seed coat color marker segregated at a ratio 15 (brown) : 1 (yellow), indicating the digenic inheritance pattern of the trait.

PCR-Based Assay for Rapid and Specific Detection of the New Xanthomonas oryzae pv. oryzae K3a Race Using an AFLP-Derived Marker

  • Song, Eun-Sung;Kim, Song-Yi;Noh, Tae-Hwan;Cho, Heejung;Chae, Soo-Cheon;Lee, Byoung-Moo
    • Journal of Microbiology and Biotechnology
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    • v.24 no.6
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    • pp.732-739
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    • 2014
  • We describe the development of a polymerase chain reaction method for the rapid, precise, and specific detection of the Xanthomonas oryzae pv. oryzae (Xoo) K3a race, the bacterial blight pathogen of rice. The specific primer set was designed to amplify a genomic locus derived from an amplified fragment length polymorphism specific for the K3a race. The 1,024 bp amplicon was generated from the DNA of 13 isolates of Xoo K3a races out of 119 isolates of other races, pathovars, and Xanthomonas species. The assay does not require isolated bacterial cells or DNA extraction. Moreover, the pathogen was quickly detected in rice leaf 2 days after inoculation with bacteria and at a distance of 8 cm from the rice leaf 5 days later. The results suggest that this PCR-based assay will be a useful and powerful tool for the detection and identification of the Xoo K3a race in rice plants as well as for early diagnosis of infection in paddy fields.

Molecular markers based on chloroplast and nuclear ribosomal DNA regions which distinguish Korean-specific ecotypes of the medicinal plant Cudrania tricuspidata Bureau

  • Lee, Soo Jin;Shin, Yong-Wook;Kim, Yun-Hee;Lee, Shin-Woo
    • Journal of Plant Biotechnology
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    • v.44 no.3
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    • pp.235-242
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    • 2017
  • Cudrania tricuspidata Bureau is a widely-used, medicinal, perennial and woody plant. Obtaining information about the genetic diversity of plant populations is highly important with regard toconservation and germplasm utilization. Although C. tricuspidata is an important medicinal plant species registered in South Korea, no molecular markers are currently available to distinguish Korean-specific ecotypes from other ecotypes from different countries. In this study, we developed single nucleotide polymorphism (SNP) markers derived from the chloroplast and nuclear genomic sequences, which serve to to identify distinct Korean-specific ecotypes of C. tricuspidata via amplification refractory mutation system (ARMS)-PCR and high resolution melting (HRM) curve analyses. We performed molecular authentication of twelve C. tricuspidata ecotypes from different regions using DNA sequences in the maturaseK (MatK) chloroplast intergenic region and nuclear ribosomal DNA internal transcribed spacer (ITS) regions. The SNP markers developed in this study are useful for rapidly identifying specific C. tricuspidata ecotypes from different regions.

Genetic Distances in Three Ascidian Species determined by PCR Technique

  • Yoon, Jong-Man
    • Development and Reproduction
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    • v.20 no.4
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    • pp.379-385
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    • 2016
  • Seven oligonucleotides primers were shown to generate the shared loci, specific loci, unique shared loci to each species and shared loci by the three species which could be obviously scored. In the present study, 7 oligonucleotides primers produced 401 total loci in the Styela clava (SC) species, 390 in the Halocynthia roretzi (HR) and 434 in the Styela plicata (SP), respectively. Seven oligonucleotides primers generated 275 specific loci in the SC, 341 in the HR and 364 in the SP species, respectively. The oligonucleotides primer BION-23 generated 28 unique loci to each species in the SP species. Especially, the oligonucleotides primer BION-25 produced 7 unique loci to each species, which were identifying each species in the SP species. BION-17 distinguished 21 shared loci by the three ascidian species, major and/or minor fragments of sizes, which were identical in almost all of the samples. Based on the average bandsharing values of all samples, the similarity matrix ranged from 0.519 to 0.774 in the SC species, from 0.261 to 0.683 in the HR species and from 0.346 to 0.730 in the SP species. As regards average bandsharing value (BS) results, individuals from SC species ($0.661{\pm}0.081$) exhibited higher bandsharing values than did individuals from HR species ($0.555{\pm}0.074$) (P<0.05). The dendrogram obtained by the seven oligonucleotides primers indicates three genetic groups. In three ascidian species, the shortest genetic distance (0.071) exhibiting significant molecular difference was also between individual no. 20 and no. 21 within the SP species.

Cryptosporidium hominis Infection Diagnosed by Real-Time PCR-RFLP

  • Cheun, Hyeng-Il;Kim, Kyungjin;Yoon, Sejoung;Lee, Won-Ja;Park, Woo-Yoon;Sim, Seobo;Yu, Jae-Ran
    • Parasites, Hosts and Diseases
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    • v.51 no.3
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    • pp.353-355
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    • 2013
  • There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections.