Journal Article

Acanthamoeba' nin Hizli Tespiti için 18S rRNA Genine Özgü Hizli Döngü Aracili Izotermal Amplifikasyon Assay for Rapid Detection of Acanthamoeba

Döskaya, Aysu Degirmenci, Akil, Mesut, Dagci, Hande, Karakavuk, Muhammet, Can, Hüseyin, Gürüz, Adnan Yüksel, Aykur, Mehmet, Döskaya, Mert

2023

Overview

Amaç: Serbest yasayan amiplerden biri olan Acanthamoeba, basta su, toprak ve hava olmak üzere birçok çevresel örnekte tespit edilmistir. Acanthamoeba'nin neden oldugu en önemli klinik tablolar içinde Acanthamoeba keratiti ve granülomatöz amibik ensefalit bulunmaktadir. Bu çalismada klinik ve çevresel örneklerde Acanthamoeba'nin varliginin daha hizli bir sekilde saptanabilmesi için Acanthamoeba 18S rRNA gen bölgesine özgü primerler ile gelistirilen hizli döngü aracili izotermal amplifikasyon (LAMP) testinin hassasiyetinin belirlenmesi amaçlanmistir. Yöntemler: Kültürde çogaltilan Acanthamoeba spp. susu 200 [micro]L'de 1x10 (6) trofozoit olacak sekilde sulandirilip DNA izolasyonu yapilarak DNA miktari Nano-Drop Spektrofotometre ile belirlenmistir. Saflastirilan DNA'lar 1000 pg'den 0,001 pg'ye kadar seyreltilip, kolorimetrik ve floresan tabanli iki farkli LAMP reaksiyonunda kullanilmistir. LAMP reaksiyon karisimi toplam 25 [micro]L hacimde 63 [degrees]C'de 60 dk olacak sekilde inkübe edilmistir. Bulgular: Gelistirilen testlerin hassasiyetinin saptanmasinda elde edilen Acanthamoeba spp. genomik DNA'sini hem kolorimetrik hem de floresan tabanli LAMP testlerinde 1, 10, 100 ve 1000 pg/reaksiyon pozitiflik gözlenmistir. Hem kolorimetrik hem de floresan LAMP testinin en düsük analitik hassasiyeti 1 pg/reaksiyon olarak tespit edildi. Ayrica, testin özgüllügünü degerlendirmek için diger parazit DNA'lari ile uygulanan LAMP reaksiyonu sonucunda kolorimetrik ve %1'lik agaroz jel elektroforezinde pozitif kontrol disinda LAMP ürünü tespit edilmemis olup, gelistirilen testin özgüllügü %100 olarak tespit edilmistir. Sonuç: Acanthamoeba'nin 18S rRNA gen bölgesi hedef alinarak gelistirilen LAMP testinin 1 pg genomik DNA saptama limitine sahip oldugu ortaya konulmustur. LAMP testinin kültür yöntemine göre daha hassas ve hizli olmasinin yani sira basit, ucuz ve yüksek hassasiyete sahip olmasi umut vericidir. Bu nedenle gelistirilen testin çevresel ve klinik örneklerde Acanthamoeba spp. tanisi için uygulanabilecegi düsünülmektedir. Anahtar Kelimeler: Acanthamoeba spp., 18S rRNA geni, hassasiyet, kolorimetrik LAMP, floresan LAMP Objective: Acanthamoeba, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. Acanthamoeba keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by Acanthamoeba. In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to Acanthamoeba 18S rRNA gene to detect more rapidly the presence of Acanthamoeba in clinical and environmental samples. Methods: Acanthamoeba strain grown in culture was diluted in 200 [micro]L as 1x10 (6) trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 [degrees]C in a total volume of 25 [micro]L. Results: To determine the sensitivity of the test, positivity of Acanthamoeba genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%. Conclusion: It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of Acanthamoeba has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of Acanthamoeba in environmental and clinical samples. Keywords: Acanthamoeba spp., 18S rRNA gene, sensitivity, colorimetric LAMP, fluorescence LAMP

• Publisher: Galenos Yayinevi Tic. Ltd
• Subject: DNA / Fluorescence / Genes / Genetic research / RNA