• Title/Summary/Keyword: universal primers

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Universal-, Genus-specific, Species-specific Probes and Primers Design for Microbial Identification

  • Park, Jun-Hyung;Park, Hee-Kyung;Song, Eunsil;Jang, Hyun-Jung;Kang, Byeong-Chul;Lee, Seung-Won;Kim, Hyun-Jin;Kim, Cheol-Min
    • Proceedings of the Korean Society for Bioinformatics Conference
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    • 2005.09a
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    • pp.399-401
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    • 2005
  • MIPROBE is a web-based tool for design of universal, genus-specific, and species-specific primers and probes. The main functions of MIPROBE are collection of target gene sequences, construction of consensus sequences, collection of candidate primers and probes, and evaluation of candidates by BLAST. Biologists with little computer skills can easily use MIPROBE to design large-scale universal, genus-, and species-specific primers and probes. This software is available at http://www.miprobe.com. Also detailed descriptions of how to use the program are found at this site.

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Development of Broad-range and Specific 16S rRNA PCR for Use in Routine Diagnostic Clinical Microbiology (임상미생물 검출을 위한 광대한 범위와 특이도를 가지는 16S rRNA PCR법 개발)

  • Kim, Hyun-Chul;Kim, Yun-Tae;Kim, Hyogyeong;Lee, Sanghoo;Lee, Kyoung-Ryul;Kim, Young-Jin
    • Journal of Life Science
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    • v.24 no.4
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    • pp.361-369
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    • 2014
  • Broad-range and specific 16S rRNA gene PCR is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. We describe the development of a broad-range and specific PCR primer, based on bacterial 16S rRNA, for use in routine diagnostic clinical microbiology services. The primers were designed by using conservative regions of 16S rRNA sequences from 10 strains. Ninety-eight clinical strains were isolated from clinical patient specimens. A total of 98 strains of bacteria were identified by phenotypic methods; PCR with newly designed primers and universal primers. All purified PCR products were sequenced using both forward and reverse primers on an automated DNA analyzer. In this study, we evaluated the usefulness of the newly designed primers and the universal primers for the detection of bacteria, and both these techniques were compared with phenotypic methods for bacteria detection. When we also tested 98 strains of clinical isolates with newly designed primers, about 778 bp DNA fragments were amplified and identified from all strains. Of the 98 strains, 94 strains (95.9%) correspond in comparison with phenotypic methods. The newly designed primers showed that the identities of 98 (100%) strains were the same as those obtained by universal PCR primers. The overall agreement between the newly designed primers and universal primers was 100%. The primer set was designed for rapid, accurate, and cheap identification of bacterial pathogens. We think the newly designed primer set is useful for the identification of pathogenic bacteria.

Random amplified polymorphic DNA analysis of bacterial pathogens using universal rice primers

  • Monoldorova, Sezim;Kim, Jinsol;Kim, Joon Hee;Jeon, Bo-Young
    • Korean Journal of Veterinary Service
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    • v.40 no.1
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    • pp.1-6
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    • 2017
  • Molecular typing of pathogenic microorganisms is important for epidemiological investigation of infectious disease outbreaks. In this study, we applied Universal Rice Primers (URP) that were originated from repetitive sequences in rice chromosomal DNA to random amplified polymorphic DNA (RAPD) analysis of pathogenic bacteria such as Escherichia coli, Listeria monocytogenes, and Salmonella sp. Of the twelve URP primers examined to date, seven primers (URP-2, -3, -4, -5, -6, -8, and -9) generated reproducible and polymorphic PCR products ranging from 1 to 13 bands. One of them, URP-6 was very effective in differentiating seven E. coli serotypes, seven L. monocytogenes clinical isolates, and eight Salmonella subspecies (ssp.) serovars. The results thus indicate that RAPD analysis using URP primers might be useful in typing bacterial pathogens including E. coli, L. monocytogenes, and Salmonella strains.

PCR Based Detection of Phellinus linteus using Specific Primers Generated from Universal Rice Primer(URP) Derived PCR Polymorphic Band

  • Kang, Hee-Wan;Park, Dong-Suk;Park, Young-Jin;Lee, Byoung-Moo;Cho, Soo-Muk;Kim, Ki-Tae;Seo, Geon-Sik;Go, Seung-Joo
    • Mycobiology
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    • v.30 no.4
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    • pp.202-207
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    • 2002
  • This study was carried out to develop specific primers for PCR detection of Phellinus linteus. Diverse genomes of 15 Phellinus spp. including five Phellinus linteus isolates were fingerprinted by Primer Universal rice primer(URP)1F. The URP-PCR pattern differentiated P. linteus isolates from other phellinus spp. A polymorphic band(2.8 kb), which is unique for P. linteus isolates, was isolated and sequenced. Twenty four-oligonucleotide primer pairs were designed based on information of DNA sequence. The primer set(PLSPF2/PLSPR1) amplified single band(2.2 kb) of expected size with genomic DNA from seven Phellinus linteus, but not with that of other Phellinus species tested. The primers could be used identically in both DNA samples from mycelium and fruit bodies. This specific primers could offer a useful tool for detecting and identifying P. linteus rapidly.

Identification of Phellinus linteus by Morphological Characteristics and Molecular Analysis (형태적.분자생물학적 방법에 의한 Phellinus linteus의 동정에 관한 연구)

  • Kim, Sang-Hee;Kim, Soo-Ho;Sung, Jae-Mo;Harrington, Thomas C.
    • The Korean Journal of Mycology
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    • v.27 no.5 s.92
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    • pp.337-340
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    • 1999
  • The context and upper surface of Phellinus basidiocarp become blackened, rimose and woody. The basidiocarp is sessile, dimidiate and elongate. The basidiospores are pigmented and ovoid to globose. Hymenial setae are $17{\sim}35{\times}6{\sim}8{\mu}m$. Nineteen isolates of Phellinus species, including Phellinus linteus, were used for sequencing of the internal transcribed spacer (ITS) region of the nuclear rDNA. Based on these sequence data, specific primers were designed for identification of Phellinus linteus isolates in Korea. The specific primers were within the ITS1 and ITS2 regions and were nested within the universal primers flanking the spacer regions. A total of four primers (the universal primers ITS-1F and ITS-4, and the specific primers PL-F and PL-R) were used for detection of Phellinus linteus collected in Korea. The length of the four amplification products of Phellinus linteus DNA were 800 bp (ITS-1F/ITS-4), two bands of about 720 bp (ITS-1F/PL-R and PL-F/ITS-4), and 610 bp (PL-F/PL-R). Among 23 isolates of Phellinus species collected in Korea, Thirteen isolates were identified as Phellinus linteus based on the presence of the four bands. The other species produced only the single ITS-1F/ITS-4 product.

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Comparison of bond strengths of ceramic brackets bonded to zirconia surfaces using different zirconia primers and a universal adhesive

  • Lee, Ji-Yeon;Ahn, Jaechan;An, Sang In;Park, Jeong-won
    • Restorative Dentistry and Endodontics
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    • v.43 no.1
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    • pp.7.1-7.7
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    • 2018
  • Objectives: The aim of this study is to compare the shear bond strengths of ceramic brackets bonded to zirconia surfaces using different zirconia primers and universal adhesive. Materials and Methods: Fifty zirconia blocks ($15{\times}15{\times}10mm$, Zpex, Tosoh Corporation) were polished with 1,000 grit sand paper and air-abraded with $50{\mu}m$ $Al_2O_3$ for 10 seconds (40 psi). They were divided into 5 groups: control (CO), Metal/Zirconia primer (MZ, Ivoclar Vivadent), Z-PRIME Plus (ZP, Bisco), Zirconia Liner (ZL, Sun Medical), and Scotchbond Universal adhesive (SU, 3M ESPE). Transbond XT Primer (used for CO, MZ, ZP, and ZL) and Transbond XT Paste was used for bracket bonding (Gemini clear ceramic brackets, 3M Unitek). After 24 hours at $37^{\circ}C$ storage, specimens underwent 2,000 thermocycles, and then, shear bond strengths were measured (1 mm/min). An adhesive remnant index (ARI) score was calculated. The data were analyzed using one-way analysis of variance and the Bonferroni test (p = 0.05). Results: Surface treatment with primers resulted in increased shear bond strength. The SU group showed the highest shear bond strength followed by the ZP, ZL, MZ, and CO groups, in that order. The median ARI scores were as follows: CO = 0, MZ = 0, ZP = 0, ZL = 0, and SU = 3 (p < 0.05). Conclusions: Within this experiment, zirconia primer can increase the shear bond strength of bracket bonding. The highest shear bond strength is observed in SU group, even when no primer is used.

Development of Species-Specific PCR Primers for the Detection of Streptococcus sobrinus

  • Kim, Sang-Gon;Yoo, So-Young;Kook, Joong-Ki
    • International Journal of Oral Biology
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    • v.35 no.1
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    • pp.21-25
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    • 2010
  • This study was undertaken to develop species-specific forward and universal reverse PCR primers for the detection of Streptococcus sobrinus. These primers target the variable regions of the 16S ribosomal RNA coding gene (rDNA) and their specificity was tested against 10 strains of S. sobrinus strains and 20 different species of oral bacteria using serial dilutions of the purified genomic DNA of S. sobrinus ATCC $33478^T$. Our data show that species-specific amplicons were obtained from all the S. sobrinus strains tested but not from other species. Both direct and nested PCR could detect as little as 400 pg and 4 fg of genomic DNA from S. sobrinus ATCC $33478^T$, respectively. This result suggests that these PCR primers are highly specific and sensitive and applicable to the detection of S. sobrinus.

PCR of Gut Contents for a Food Web Study of a Marine Ecosystem

  • Kim, Nack-Keun;Kim, Kyoung-Sun;Kim, Hyun-Woo
    • Fisheries and Aquatic Sciences
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    • v.10 no.4
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    • pp.179-185
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    • 2007
  • Understanding dietary habits is one of the most important factors in studying food webs and other ecological processes. Here we designed universal primers to amplify portions of the 18S and 28S rDNA sequences to examine gut contents using PCR techniques. The gut contents of sailfin sandfish (Arctoscopus japonicus) and pacific squid (Todarodes pacificus) were examined. In total, 11 families of prey were identified with 18S and 28S rDNA using the universal primers. The DNA sequence data indicated that the primer sets successfully amplified a wide spectrum of species and represented gut contents in a relatively convenient way. We found that information in the NCBI database was not yet sufficient to discriminate the species we isolated. In addition, technology for the separation of heterogeneous PCR products and better resolution and quantification protocols would help increase data accuracy.

Genetic Divesity Analysis of Fungal Species by Universal Rice Primer (URP)-PCR (Universal Rice Primer (URP)-PCR에 의한 곰팡이 종의 유전적 다양성 검정)

  • Kang, Hee-Wan
    • The Korean Journal of Mycology
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    • v.40 no.2
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    • pp.78-85
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    • 2012
  • URP primers that were derived from repetitive DNA sequence of rice weedy rice have been applied for producing PCR polymorphisms in different fungal species. URP-PCR protocol employed stringent PCR with high annealing temperature over $55^{\circ}C$ throughout the thermo-cycling reaction, giving high reproducibility. Under the PCR condition, Each single URP primer produced characteristic fingerprints from diverse genomes of different fungal species, indicating its universal applicability. URP-PCR has been accessed for applicability to various fungal species with 33 genus, 142 species and 1,489 isolates. Numerous related papers have demonstrated that URP-PCR profiles of fungal species are very useful for identifying fungal species at intra and inter species levels. The results were reviewed in this paper.

Development of PCR Assay for Identification of Buffalo Meat

  • Rajapaksha, W.R.A.K.J.S.;Thilakaratne, I.D.S.I.P.;Chandrasiri, A.D.N.;Niroshan, T.D.
    • Asian-Australasian Journal of Animal Sciences
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    • v.16 no.7
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    • pp.1046-1048
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    • 2003
  • A polymerase chain reaction (PCR) assay was developed to differentiate buffalo meat from the meat of Ceylon spotted deer (Axis axis ceylonensis), Ceylon sambhur (Cervus unicolor unicolor), cattle (Bovine), goat (Caprine), pig (Porcine), and sheep (Ovine). A set of primers were designed according to the sequence of the mitochondrial cytochrome b gene of bubalus bubalis and by PCR amplification a band of approximately 242 bp band was observed with buffalo DNA. These primers did not cross-react with DNA of other animal species tested in the study under the specified reaction conditions. A band of 649 bp was observed for all animal species tested when DNA was amplified with the universal primers indicating the presence of mitochondrial DNA in the samples. The technique was sensitive enough to identify rotten (10 days post slaughter), dried and cooked buffalo meat. The absence of a cross reaction with human DNA using the buffalo specific primers eliminates possible false positive reactions.