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首页> 外文期刊>Journal of Clinical Microbiology >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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Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge

机译：亚型18S核糖体DNA PCR的应用和测序对角膜炎患者和污水污泥中棘阿米巴的属和基因型鉴定

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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-bpAcanthamoeba-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.
机译：这项研究从18S rDNA中鉴定了亚基因PCR扩增子，这些扩增子具有（i）对棘阿米巴属（Acanthamoeba ）高度特异性，（ii）可从所有已知基因型中获得，以及（iii）可用于鉴定单个基因型。用引物JDP1和JDP2获得的423-551bp的棘阿米巴特异扩增子ASA.S1对于目的i和ii是最可靠的。该扩增子内的可变区也鉴定了基因型簇，但目的Ⅲ是通过对基因型特异性扩增子GTSA.B1进行测序而最佳实现的。由于这种扩增子可以从任何真核生物中获得，因此研究需要阿森纳 Acanthamoeba 培养物。用引物CRN5和1137产生的GTSA.B1在参考bp 1和1475之间延伸。基因型鉴定依赖于三个片段：bp 178至355、705至926和1175至1379。ASA.S1是从单个变形虫获得的。所有已知的18S rDNA基因型的细菌培养物，以及疑似 Acanthamoeba 角膜炎（AK）的苏格兰患者的角膜刮取术。 AK PCR结果与15个培养阳性标本中的11个的培养结果一致，并且在9个培养阴性标本之一中检测到 Acanthamoeba 。检测了11种培养阳性分离物中的6种的ASA.S1序列，它们与基因型簇T3-T4-T11最密切相关。使用GTSA.B1序列进行的相似距离分析将9个与南非AK相关的分离株鉴定为基因型T4，并将三个污泥中的3个分离株鉴定为基因型T5。我们的结果证明了18S核糖体DNA PCR扩增子ASA.S1和GTSA.B1分别用于 Acanthamoeba 特异性检测和可靠的基因分型，并提供了进一步的证据证明T4是AK的主要基因型。

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