Zeynep Ceren KARAHAN, Alper TEKELİ, Figen ATALAY, Nejat AKAR

Mycobacterium tuberculosis suşlarının polimeraz zincir reaksiyonu (PZR)

Amaç: Tüberküloz, günümüzde giderek artan bir toplum sağlığı sorunu haline gelmiştir. İnfeksiyon dinamiklerini incelemek ve uygun tedavi ve infeksiyon kontrol stratejileri belirleyebilmek için etkenin moleküler tiplendirmesi önemlidir. Mycobacterium tuberculosis genotiplemesinde altın standart IS6110 RFLP analizi olmakla birlikte, yöntemle ilgili zorluklar nedeniyle son yıllarda Polimeraz Zincir Reaksiyonuna (PZR) dayalı yöntemler de kullanılmaktadır. Bu çalışmada, akciğer tüberkülozlu 39 farklı hastadan izole edilen M. tuberculosis izolatının, PZR yöntemi ile genotiplendirilmesi amaçlanmıştır. Gereç ve Yöntem: Löwenstein-Jensen besiyerinde üretilen suşların hücre duvarları cam boncuklarla parçalanarak DNA elde edilmiştir. PZR ile genotiplendirme için, IS6110 insersiyon dizisinin iki ucuna yönelik hazırlanan IS-1 (5'CAA GAA CCT TTC CTA CCC CA 3') ve IS-2 (5'GGC TGA GGT CTC AGA TCA G 3') primerleri kullanılmıştır. Elde edilen bant paternleri bilgisayar ortamına aktarılarak, suşlar arası genetik yakınlık, Syngene Gene Directory 1.02.0 (Syngene, Cambridge, İngiltere) programında UPGMA metodu ile Dice katsayısı kullanılarak belirlenmiştir. Bulgular ve Sonuç: Çalışma sonucunda izolatların hepsinin genotipik olarak birbirinden farklı olduğu tespit edilmiş, yöntem kolay uygulanabilir, hızlı, ucuz ve tekrarlanabilir bulunmuştur.

Genotyping Mycobacterium tuberculosis strains with polymerase chain reaction (PCR)

Aim: Tuberculosis is an increasing public health problem world wide. In order to evaluate the infection dynamics and to develop appropriate therapeutic and infection control strategies, molecular typing of the infectious agent is important. The gold standard of genotyping Mycobacterium tuberculosis isolates is IS6110 RFLP analyses. As this method is technically hard to perform, methods relying on Polymerase Chain Reaction (PCR) typing have been increasingly used. In this study we aimed to genotype 39 M. tuberculosis isolates obtained from different pulmonary tuberculosis patients by PCR. Materials and Methods: The cell walls of the isolates grown on Löwenstein-Jensen medium were destructed by glass-beads in order to obtain genomic DNA. PCR genotyping was performed by using the primers IS-1 (5'CAA GAA CCT TTC CTA CCC CA 3') and IS-2 (5'GGC TGA GGT CTC AGA TCA G 3'), designed to cover the two sides of IS6110 insertion sequence. The band patterns were transferred to computer and genetic relatedness of the strains were analyzed by using the Syngene Gene Directory 1.02.0 (Syngene, Cambridge, England) program with UPGMA method by using the Dice coefficient. Results and Conclusion: All the isolates were found to be genetically unrelated, and the method easy to perform, rapid, cheap and reproducible.

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