Serkan BAKIRCI, Hasan EREN, Tülin KARAGENÇ, Hüseyin Bilgin BİLGİÇ, William WEIR

Theileria annulata DNA'sının loop aracılı izotermal yöntemle (LAMP) çogaltılması

Son yıllarda polimeraz zincir reaksiyonuna (PZR) alternatif olarak termal siklus kullanmadan izotermal kosullar altında hedef bölgelerin çogaltılmasına olanak saglayan LAMP (loop mediated isothermal amplification) gibi yeni teknikler gelistirilmistir. LAMP, yüksek özgünlükte ve kısa zamanda çok sayıda hedef DNA kalıbının çogaltılabildiği ve elde edilen ürünün kolay ve hızlı tespitine olanak veren bir yöntemdir. Bu çalışmada, Theileria annulata'nın merozoit yüzey antijeni (Mero1), 30 kDa major merozoit yüzey antijeni (Tams-1) ve sitokrom b genlerini çoğaltmak için özgül olarak tasarlanan primer çiftleri kullanılarak, hasta ve tasıyıcı sığırlarda T. annulata'nın tespitinde LAMP yönteminin özgüllük ve duyarlılığı degerlendirilmistir. En yüksek duyarlılıga sahip olan primer çiftleri ile LAMP'ın sahadan toplanan örneklerde uygulanabilirligi arastırılmıstır. Sitokrom b genine özgü iki primer çiftinin (CYTOB1 ile CYTOB341) T. annulata'nın farklı izolatlarını özgül olarak çogalttığı ve BL20 ve diğer türlere ait DNA'larda ise herhangi bir çogalma olmadığı belirlenmiştir. CYTOB1 primerleri 2 fg'a kadar T. annulata Ankara / D7 DNA'sını tespit edebilmiş, bununla birlikte CYTOB341'in duyarlılığı CYTOB1'ye oranla 10 kat düşük bulunmuştur. Yapılan analizlerde, CYTOB1 LAMP'ının duyarlılığı F3/B3 PZR ile aynı bulunmus, ancak CYTOB1 F3/B3 PZR'in duyarlılığının cytob1 PZR'ye oranla 10 kat daha az oldugu belirlenmistir. Ayrıca, LAMP ürünlerinin özgüllükleri restriksiyon enzimi ile keserek ve sekans analizleri ile doğrulanmıştır. Elde edilen sonuçlar, sitokrom b geni dışında hedef gen bölgelerine (Tams-1 ve Mero1) özgü tasarlanan primer çiftlerinin hiçbirinin T. annulata'nın farklı izolatlarını özgül ve duyarlı olarak tespit etmediğini göstermiştir. Sonuç olarak, CYTOB1 LAMP yönteminin T. annulata'nın saha şartlarında tespitinde kullanılabilirliginin cytob1 PZR'ye oranla düsük oldugu saptanmıştır.

Loop mediated isothermal amplification (LAMP) of Theileria annulata DNA

In the past three decades, as an alternative to PCR (polymerase chain reaction) new diagnostic techniques like LAMP (loop mediated isothermal amplification) whereby target DNA can be amplified under isothermal conditions without using thermocycler have been developed. The LAMP method allows the synthesis of large amounts of DNA in a short time with high specificity and rapid and easy detection of generated products. In this study, specificity and sensitivity of LAMP method was evaluated for the detection of T. annulata in acute infected and/or carriers cattle using primer pair specifically designed to amplify merozoite surface antigen gene (Mero1), 30 kDa major merozoite surface antigen gene (Tams-1) and cytochrome b gene of T.annulata. Primer pairs with highest sensitivity were used to evaluate the applicability of LAMP to the field samples. Two LAMP primers (CYTOB1 and CYTOB341) targeting cytochrome b gene specifically amplified DNA of different T. annulata isolates successfully while no amplification was seen in other species DNAs and BL20. CYTOB1 primers detected T. annulata Ankara / D7 DNA up to 2 fg, however the detection limit of CYTOB341 was 10 fold lower. The sensitivity of CYTOB1 LAMP assay was same with F3/B3 PCR, however when compared with that of cytob1 PCR a 10fold lower sensitivity was found. The LAMP product was confirmed by restriction digestion and sequencing. Results obtained from this study indicated that none of the designed primer pairs specific to target genes (Tams-1 and Mero1), except cytochrome b gene was able to specifically and sensitively detect different isolates of T. annulata. Consequently, it was shown that LAMP method using CYTOB1 primers is less effective than the cytob1 PCR in terms of detecting T. annulata in the field samples.

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