Identification of <i>Acanthamoeba</i> at the Generic and Specific Levels Using the Polymerase Chain Reaction

Vodkin, Michael H.; Howe, Daniel K.; Visvesvara, Govinda S.; McLaughlin, Gerald L.

doi:10.1111/j.1550-7408.1992.tb01467.x

Cited by 71 publications

(51 citation statements)

References 25 publications

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“…JDP2 is a modification of ACARNA. 1383 (40) and RGPG (11). The JDP1-JDP2 primer pair previously was shown at OSU to produce the amplimer now designated ASA.S1 from acanthamoebae isolated from tissues of freshwater fishes (10).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2001

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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 culturenegative 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.The demonstrated pathogenicity for humans and animals of organisms belonging to the genus Acanthamoeba (17, 26), coupled with the difficulty of using morphological criteria for subgeneric identification of isolates (30,38), has stimulated a number of laboratories to pursue molecular methods for detection and identification. The objective is to develop methods that are suitable for both clinical and environmental applications. The identification of amoebic isolates should be very reliable and, at least for clinical use, the detection system should be very sensitive. Several research groups, including our own, have demonstrated the usefulness of PCR methods for detection of acanthamoebae (10,15,21,25,27,40). As few as 1 to 10 trophozoites can be detected. It also is possible to enhance detection of individual amoeba in very dilute liquid clinical samples with fluorescent in situ hybridization (FISH) (36). Several molecular approaches increase the reliability of specimen identification, but the use of DNA sequence variation appears to be the most promising. The variation is observed in restriction fragment length polymorphisms of complete or partial nuclear 18S rRNA genes (8,20,21,22), of complete mitochondrial 16S rRNA genes (7, 46), and of the complete mitochondrial genome (3,7,13,18,22,45). It also is observed in the DNA seq...

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Section: Methodsmentioning

confidence: 99%

“…The identification of amoebic isolates should be very reliable and, at least for clinical use, the detection system should be very sensitive. Several research groups, including our own, have demonstrated the usefulness of PCR methods for detection of acanthamoebae (10,15,21,25,27,40). As few as 1 to 10 trophozoites can be detected.…”

mentioning

confidence: 99%

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

et al. 2001

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“…Approaches that have been useful include RFLP analysis of the entire mitochondrial genome, 12,20-25 riboprinting (described below) of 18S rDNA, 12,26 production of typespecific PCR amplimers, [27][28][29][30] analyses based on sequences of subgenic PCR amplimers [30][31][32] as well as the development of fluorescent oligonucleotide probes for in situ staining that are specific either for the genus Acanthamoeba or for genotype T4. 33 The recent introduction of a 'reverse dot-blot' technique holds promise for the eventual simultaneous detection and identification of all rDNA genotypes present in any specimen.…”

Section: Molecular Epidemiologymentioning

confidence: 99%

Acanthamoeba keratitis update—incidence, molecular epidemiology and new drugs for treatment

Seal

2003

Eye

175

135

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“…Usefulness of PCR as an sensitive and specific diagnostic tool for the demonstration of Acanthamoeba has been proved by several studies. Vodkin et al, (12) were the first to use PCR for the genusspecific detection of Acanthamoeba targeting the 18S rDNA using the primer pair ACARNA f1383 and ACARNA r1655.…”

Section: Resultsmentioning

confidence: 99%

Utility of Polymerase Chain Reaction in Diagnosis of Acanthamoebaand Microsporidial Keratitis

Bhosale¹,

Parija²,

Mandal³

et al. 2016

Int.J.Curr.Microbiol.App.Sci

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Identification of Acanthamoeba at the Generic and Specific Levels Using the Polymerase Chain Reaction

Cited by 71 publications

References 25 publications

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

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

Acanthamoeba keratitis update—incidence, molecular epidemiology and new drugs for treatment

Utility of Polymerase Chain Reaction in Diagnosis of Acanthamoebaand Microsporidial Keratitis

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