Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge
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| Published in | Journal of Clinical Microbiology Vol. 39; no. 5; pp. 1903 - 1911 |
|---|---|
| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | English |
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Washington, DC
American Society for Microbiology
01.05.2001
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| Online Access | Get full text |
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| AbstractList | This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK. This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba , (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba -specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba -specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK. Article Usage Stats Services JCM Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter current issue JCM About JCM Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy JCM RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0095-1137 Online ISSN: 1098-660X Copyright © 2014 by the American Society for Microbiology. For an alternate route to JCM .asm.org, visit: JCM |
| Author | David V. Seal Ingrid A. Niszl Jill M. Schroeder Thomas J. Byers Paul A. Fuerst Gregory C. Booton John Hay Miles B. Markus |
| AuthorAffiliation | Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210 1 ; Tennent Institute of Ophthalmology, Western Infirmary, Glasgow University, Glasgow, United Kingdom 2 ; and Parasitology Research Program, University of the Witwatersrand, Johannesburg, WITS, 2050, South Africa 3 |
| AuthorAffiliation_xml | – name: Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210 1 ; Tennent Institute of Ophthalmology, Western Infirmary, Glasgow University, Glasgow, United Kingdom 2 ; and Parasitology Research Program, University of the Witwatersrand, Johannesburg, WITS, 2050, South Africa 3 |
| Author_xml | – sequence: 1 givenname: Jill M surname: SCHROEDER fullname: SCHROEDER, Jill M organization: Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, United States – sequence: 2 givenname: Gregory C surname: BOOTON fullname: BOOTON, Gregory C organization: Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, United States – sequence: 3 givenname: John surname: HAY fullname: HAY, John organization: Tennent Institute of Ophthalmology, Western Infirmary, Glasgow University, Glasgow, United Kingdom – sequence: 4 givenname: Ingrid A surname: NISZL fullname: NISZL, Ingrid A organization: Parasitology Research Program, University of the Witwatersrand, Johannesburg, WITS, 2050, South Africa – sequence: 5 givenname: David V surname: SEAL fullname: SEAL, David V organization: Tennent Institute of Ophthalmology, Western Infirmary, Glasgow University, Glasgow, United Kingdom – sequence: 6 givenname: Miles B surname: MARKUS fullname: MARKUS, Miles B organization: Parasitology Research Program, University of the Witwatersrand, Johannesburg, WITS, 2050, South Africa – sequence: 7 givenname: Paul A surname: FUERST fullname: FUERST, Paul A organization: Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, United States – sequence: 8 givenname: Thomas J surname: BYERS fullname: BYERS, Thomas J organization: Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, United States |
| BackLink | http://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1100255$$DView record in Pascal Francis https://www.ncbi.nlm.nih.gov/pubmed/11326011$$D View this record in MEDLINE/PubMed |
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| Cites_doi | 10.1111/j.1550-7408.1992.tb01467.x 10.2307/3285675 10.1111/j.1750-3639.1997.tb01076.x 10.1128/JCM.38.11.3932-3936.2000 10.1111/j.1550-7408.1980.tb04684.x 10.3347/kjp.1996.34.4.259 10.1111/j.1550-7408.1990.tb01141.x 10.1111/j.1550-7408.1998.tb05068.x 10.1111/j.1550-7408.1993.tb04943.x 10.3347/kjp.1996.34.1.79 10.1016/0378-1119(86)90043-0 10.1128/AEM.66.10.4408-4413.2000 10.3347/kjp.1999.37.3.181 10.1128/jcm.32.4.1070-1073.1994 10.1007/s002940050368 10.1590/S0100-879X2000000100003 10.2307/3284628 10.1093/clind/13.Supplement_5.S369 10.3347/kjp.1998.36.2.69 10.1016/B978-0-12-426013-9.50009-X 10.1093/nar/25.24.4876 10.1111/j.1550-7408.1990.tb01267.x 10.1016/S0074-7696(08)61430-8 10.1007/s004360050259 10.1128/JCM.37.8.2687-2693.1999 10.1111/j.1550-7408.1969.tb02329.x 10.1016/0166-6851(94)00049-S 10.1016/S0723-2020(11)80448-0 10.1007/BF00539451 10.1093/nar/22.4.592 10.3347/kjp.1996.34.2.127 10.1093/nar/17.21.8543 10.1128/jcm.29.2.310-314.1991 10.3347/kjp.1995.33.4.331 10.1016/S1367-0484(99)80003-4 10.1001/archopht.118.2.178 |
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| Notes | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 Present address: 336 Glagow Rd., Ralston, Paisley, PA1 3BH, Scotland, United Kingdom. Present address: Department of Optometry and Vision Science, City University, London EC1 V7DD, England. Corresponding author. Mailing address: Department of Molecular Genetics, The Ohio State University, 484 W. 12th Ave., Columbus, OH 43210-1292. Phone: (614) 292-5963. Fax: (614) 292-4466. E-mail: byers.2@osu.edu. Present address: Department of Biochemistry, Indiana University School of Medicine, Indianapolis, IN 46202. |
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Mendeley... This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes,... This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba , (ii) obtainable from all known genotypes,... |
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| SubjectTerms | Acanthamoeba Acanthamoeba - classification Acanthamoeba - genetics Acanthamoeba - isolation & purification Acanthamoeba Keratitis - parasitology Amibiasis Animals Biological and medical sciences Cornea - parasitology DNA Primers DNA, Protozoan - analysis DNA, Protozoan - genetics DNA, Ribosomal - analysis DNA, Ribosomal - genetics Fundamental and applied biological sciences. Psychology General aspects and techniques Genotype Human protozoal diseases Humans Infectious diseases Medical sciences Molecular Sequence Data Parasitic diseases Parasitology Phylogeny Polymerase Chain Reaction - methods Protozoa Protozoal diseases RNA, Ribosomal, 18S - genetics rRNA 18S Sensitivity and Specificity Sequence Analysis, DNA Sewage - parasitology |
| Title | Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge |
| URI | http://jcm.asm.org/content/39/5/1903.abstract https://www.ncbi.nlm.nih.gov/pubmed/11326011 https://search.proquest.com/docview/17886586 https://pubmed.ncbi.nlm.nih.gov/PMC88046 |
| Volume | 39 |
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