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Journal of Clinical Microbiology, May 2001, p. 1903-1911, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1903-1911.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge

Jill M. Schroeder,1,dagger Gregory C. Booton,1 John Hay,2,Dagger Ingrid A. Niszl,3 David V. Seal,2,§ Miles B. Markus,3 Paul A. Fuerst,1 and Thomas J. Byers1,*

Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 432101; Tennent Institute of Ophthalmology, Western Infirmary, Glasgow University, Glasgow, United Kingdom2; and Parasitology Research Program, University of the Witwatersrand, Johannesburg, WITS, 2050, South Africa3

Received 19 October 2000/Returned for modification 23 January 2001/Accepted 26 February 2001

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.

* 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{at}osu.edu.

dagger Present address: Department of Biochemistry, Indiana University School of Medicine, Indianapolis, IN 46202.

Dagger 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.

Journal of Clinical Microbiology, May 2001, p. 1903-1911, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1903-1911.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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