Mert DÖŞKAYA, Muhammet KARAKAVUK, Hüseyin CAN, Cemal ÜN, Tuğba KARAKAVUK, Ceren GÜL, Sedef ERKUNT ALAK, Aytül GÜL, Aysu DEĞİRMENCİ DÖŞKAYA, Adnan Yüksel GÜRÜZ

Toxoplasma gondii 529 baz çifti büyüklüğünde tekrar bölgesine (RE) özgü hızlı döngü aracılı izotermal amplifikasyon testinin geliştirilmesi ve analitik hassasiyetinin belirlenmesi

Amaç: Toxoplasma gondii insan ve sıcakkanlı hayvanlarda hastalıklara neden olan bir protozoondur.Bu çalışmanın amacı toksoplazmozis tanısı için T. gondii tekrar bölgesine (RE) özgü primerlerintasarlanması ile geliştirilen floresans esaslı hızlı Döngü Aracılı İzotermal Amplifikasyon (LAMP) testininanalitik hassasiyetini belirlemektir.Gereç ve Yöntem: Polimeraz Zincir Reaksiyonu (PZR) ile elde edilen T. gondii RE, pCR2.1-TOPOvektörüne klonlanmıştır. Spesifik primerler kullanılarak geliştirilen LAMP testinin analitik hassasiyetipCR2.1-RE vektörünün seri seyreltmeleri ile belirlenmiştir.Bulgular: T. gondii RE genine özgü tasarlanan primerler ile geliştirilen LAMP testinin analitikhassasiyetinin ≤10 plazmit/reaksiyon olduğu tespit edilmiştir.Sonuç: RE bölgesi T. gondii geni içinde 200-300 kere tekrar ettiğinden dolayı geliştirilen testin 0,05takizoit/reaksiyon saptama limitine sahip olduğu hesaplanmıştır. LAMP testinin kolay, ucuz, hızlı vesaha koşullarına uygun olması

Development of toxoplasma gondii 529 base pair size repeat region (RE) specific rapid loop mediated isothermal amplification test and determination of analytical sensitivity

Aim: Toxoplasma gondii is a protozoon that causes disease in humans and warm-blooded animals. The aim of this study is to determine the analytical sensitivity of the fluorescence-based rapid loop mediated isothermal amplification (LAMP) test developed by designing primers specific to T. gondii repeat region (RE) for the diagnosis of toxoplasmosis. Materials and Methods: T. gondii RE obtained with PCR was cloned into vector pCR2.1-TOPO. The analytical sensitivity of the LAMP assay developed using specific primers was determined by serial dilutions of the pCR2.1-RE vector Results: It was determined that the analytical sensitivity of the LAMP test developed with primers designed specific to T. gondii RE gene was 10 copy plasmid / reaction. Conclusion: Since the RE region repeats 200-300 times in the T. gondii gene, the test developed has been calculated to have a detection limit of 0.05 tachyzoites/reaction. The LAMP test is predicted to be promising because it is easy, cheap, fast and suitable for field conditions and is more sensitive than other molecular tests.

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