Ehrlichia spp. disülfid oksidoredüktaz genini hedefleyen polimeraz zincir reaksiyonunun analitik duyarlılığının belirlenmesi: Klinik olarak leishmaniasis'ten şüphelenilen bir köpekte ehrlichiosis'in moleküler tanısı

Öz

Ehrlichia spp. insan ve hayvanları enfekte edebilen kene kaynaklı zoonotik patojenlerdir. Günümüzde hastalığın tanısında kullanılan testler arasında yüksek duyarlılık ve özgünlüğe sahip olan moleküler yöntemlerin önemi giderek artmaktadır. Bu çalışmanın amacı Ehrlichia spp. disülfid oksiredüktaz (DSB) genini hedefleyen konvansiyonel polimeraz zincir reaksiyonunun (PZR) analitik hassasiyetinin belirlenmesidir. Öncelikle Ehrlichia spp. DSB geni TOPO vektörüne klonlanmıştır. DSB geni içeren TOPO plazmidi seri olarak sulandırıldıktan sonra Ehrlichia spp. DSB genini hedefleyen PCR gerçekleştirilmiştir. Bu araştırma üzerinde çalışmalar sürerken, klinik olarak leishmaniasis şüphesi bulunan ve leishmaniasis tedavisi alan bir sokak köpeğinin kan ve deri kazıntı örnekleri laboratuvarımıza gelmiştir. Sokak köpeğine ait kan ve deri kazıntı örneklerinde patojeni belirlemek amacıyla Ehrlichia spp. DSB ve 16S rRNA ile Leishmania spp. kinetoplast DNA (kDNA) genlerini hedef alan PCR testleri yapılmıştır. DSB geni içeren seri sulandırılmış TOPO plazmidi kullanılarak yapılan Ehrlichia spp. DSB genini hedefleyen PCR testinin analitik hassasiyeti, 1 ≥ kopya plazmit/reaksiyon olarak tespit edilmiştir. Klinik olarak leishmaniasis şüphesi bulunan sokak köpeğinin kan ve deri kazıntı örneklerinde Ehrlichia spp. DSB genini hedefleyen PCR pozitif, Leishmania spp. kDNA'yı hedefleyen PCR testleri negatif olarak tespit edilmiştir. Ehrlichia spp. 16S rRNA'yı hedefleyen nested PCR testi ile, sokak köpeğinin kan ve deri kazıntı örneklerinde E. canis olarak tanımlanmıştır. Bu çalışmada Ehrlichia spp. DSB genini hedefleyen PCR'nin yüksek duyarlılığa sahip olduğu gösterilmiştir. Ayrıca moleküler yöntemlerin klinisyenlere ehrlichiosis ve leishmaniasis ayırıcı tanısında uygunsuz tedaviyi önlemede yardımcı olabileceği öngörülmüştür.

Anahtar Kelimeler

• Disülfit oksiredüktaz
• Ehrlichia canis
• Ehrlichiosis
• PCR
• Leishmaniasis

Determining the analytical sensitivity of polymerase chain reaction targeting Ehrlichia spp. disulfide oxidoreductase gene: Molecular diagnosis of ehrlichiosis in a dog clinically suspected with leishmaniasis

Abstract

Ehrlichia spp. is tick-borne zoonotic pathogen that can infect humans and animals. Nowadays, among the tests used in the diagnosis of ehrlichiosis, the importance of molecular methods is increasing steadily due to their high sensitivity and specificity. The aim of this study was to determine the analytical sensitivity of a conventional polymerase chain reaction (PCR) targeting Ehrlichia spp. disulfide oxidoreductase (DSB) gene. Ehrlichia spp. DSB gene was cloned into the TOPO vector. After TOPO plasmid containing DSB gene were serially diluted, PCR targeting the Ehrlichia spp. DSB gene was performed. While working on this research, blood and skin scraping samples of a stray dog clinically suspected with leishmaniasis as well as treated for leishmaniasis arrived to our laboratory. Thereafter, PCRs targeting Ehrlichia spp. DSB and 16S rRNA and Leishmania kinetoplast DNA (kDNA) genes were performed to identify the pathogen in blood and skin scraping samples of the stray dog. The analytical sensitivity of the PCR assay targeting Ehrlichia spp. DSB gene was 1 ≥ copy plasmid/reaction using serially diluted TOPO plasmid containing DSB gene. PCR targeting the Ehrlichia spp. DSB gene was positive and PCR targeting Leishmania spp. kDNA was negative in blood and skin samples of the stray dog clinically suspected with leishmaniasis. Using nested PCR targeting Ehrlichia spp. 16S rRNA, E. canis was identified in blood and skin scraping samples of the stray dog. In this study, PCR targeting Ehrlichia spp. DSB gene has been shown to have high sensitivity. Also it was shown molecular methods can help clinicians in differential diagnosis of ehrlichiosis and leishmaniasis to prevent inappropriate treatment.

Keywords

• Disulfide oxidoreductase
• Ehrlichia canis
• Ehrlichiosis
• PCR
• Leishmaniasis

Kaynakça

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