FARKLI SU KAYNAKLARINDAN İZOLE EDİLEN ACANTHAMOEBA TÜRLERİNİN MOLEKÜLER PREVALANSI VE GENOTİPLERİNİN BELİRLENMESİ

Bu çalışma, farklı su kaynaklarından elde edilmiş Acanthamoeba türlerinin moleküler prevalanslarının saptanması ve 18S rRNA gen bölgesine göre filogenetik karakterlerinin belirlenmesi amacıyla gerçekleştirilmiştir. Çalışmada, Sinop ve Ordu yörelerindeki çeşme suyu, havuz suları, kaplıca ve göllerden 80 örnek toplanmıştır. Su örneklerinin kültür ortamında üretilmesini takiben, DNA izolasyonu ve PCR analizleri gerçekleştirilmiştir. 18S rRNA gen bölgesi yönünden pozitif belirlenen amplikonlar klonlanmış ve plazmid pürifikasyonu yapılmıştır. Plazmidler vektör spesifik primerlerle sekanslanarak hedef dizilimler elde edilmiştir. İlgili sekanslarla birlikte GenBank veri tabanında kayıtlı benzer izolatları içeren toplam 31 sekanslık veri seti oluşturulmuştur. Çalışmada, Sinop yöresinde %17,1, Ordu yöresinden %20 Acanthamoeba pozitifliği tespit edilmiştir. Filogenetik incelemelerde, elde edilen Acanthamoeba izolatlarının Türkiye’den ve dünyadan rapor edilen T4 genotipine ait izolatlarla aynı kümede kümelendikleri görülmüştür. İzolatlar arasında iki haplotip saptanmış ve ortalama haplotip diversitesi 0.682±0.084 olarak belirlenmiştir. 18S rRNA veri setinde, TRERUAcantha1 ve TRERUAcantha2 haplotiplerinin de bulunduğu T4 genotipindeki tüm izolatların %100 identik oldukları belirlenmiştir. Filogenetik analizlerde T4 genotipindeki izolatların monofiletik yapılanma gösterdiği saptanmıştır. T4 genotipinde oldukları saptanan izolatlarımızın %99.9 ile Almanya’da kontakt lensten izole edilen ve T13 genotipinde yer alan KaBo (KJ476522) izolatıyla en yakın benzerliği gösterdiği görülmüştür.

Anahtar Kelimeler:

18S rRNA, Acanthamoeba, genotiplendirme, moleküler prevalans, su örneği, genotyping, molecular prevalence, water sample., water sample

MOLECULAR PREVALANCE AND GENOTYPING OF ACANTAHAMOEBA SPECIES ISOLATED FROM VARIOUS WATER SUPPLIES

This study was conducted to determine the molecular prevalence of Acanthamoeba species isolated from various water sources and to determine their molecular characteristics for the 18S rRNA gene region. In this study, a total of 80 samples were collected from fountain water, pool water, hot springs and lakes in Sinop and Ordu provinces. Following the production of water samples in the culture medium, DNA isolation and PCR analyzes were performed. Amplicons determined positive for the 18S rRNA gene region were cloned and plasmid purification was performed. Target sequences were obtained by sequencing the plasmids with vector-specific primers. A total of 31 sequence data sets were created, containing similar isolates registered in the Gen Bank database, together with the relevant sequences. In the study, 17.1% Acanthamoeba positivity was found in the Sinop region and 20% in the Ordu region. In phylogenetic analyses, it has been shown that the obtained Acanthamoeba isolates clustered in the same cluster with the T4 genotype isolates from Turkey and the world. Two haplotypes were detected among the positive isolates and the mean haplotype diversity was 0.682±0.084. In the 18S rRNA dataset, all isolates in the T4 genotype, including the TRERUAcantha1 and TRERUAcantha2 haplotypes, were 100% identical. In phylogenetic analyzes, isolates in the T4 genotype showed a monophyletic structure. It was observed that our isolates found to be T4 genotype showed 99.9% identity with KaBo (KJ476522) isolate identity, which was isolated from contact lenses in Germany and included in the T13 genotype.

Keywords:

18S rRNA, genotyping, Acanthamoeba, molecular prevalence, water sample,

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