ALPARSLAN YILDIRIM, ÖNDER DÜZLÜ, ABDULLAH İNCİ, ZUHAL ÖNDER, ARİF ÇİLOĞLU

Sığırlarda Babesia bovis ve Babesia bigemina'nın Reverse Line Blotting, Nested PCR ve real time PCR teknikleri ile karşılaştırmalı tanısı

Bu çalışma, sığırlarda Babesia bovis ve Babesia bigeminanın moleküler teşhisinde Reverse Line Blotting (RLB), Nested PCR ve Real Time PCR tekniklerinin kıyaslanması amacıyla planlanmıştır. Türkiyenin farklı illerindeki sığırlardan daha önce farklı projelerde kullanılmak üzere toplanmış ve laboratuarda muhafaza edilen 400 adet kan örneğinden genomik DNA ekstraksiyonu yapılmıştır. Elde edilen genomik DNAların konsantrasyonları NanoDrop spektrofotometrede ölçülmüş ve uygun konsantrasyonları hazırlandıktan sonra RLB, Nested PCR ve Real Time PCR teknikleri ile analiz edilmiştir. RLB sonuçlarına göre incelenen örneklerin toplam 18inin (%4.50) B. bovis, 59unun (%14.75) B. bigemina, 16sının (%4.00) Babesia spp. ve 2sinin (%0.50) B. bovis + B. bigemina; Nested PCR ile 23ünün (%5.75) B. bovis, 71inin (%17.75) B. bigemina ve 7sinin (%1.75) B. bovis + B. bigemina; Real Time PCR ile 23ünün (%5.75) B. bovis, 75inin (%18.75) B. bigemina ve 9unun (%2.00) ise B. bovis + B. bigemina ile miks enfekte olduğu belirlenmiştir. Real Time PCR tekniği ile kıyaslanması sonucu Nested PCR tekniğinin %94.4 sensitivite ve %100.0 spesifite gösterdiği; RLB tekniğinin ise %88.8 sensitivite ve %100.0 spesifiteye sahip olduğu belirlenmiştir. RLB testinde Babesia spp. belirlenen 16 örneğin hem Real Time PCR hem de Nested PCRda 5inin B. bigemina, 9unun B. bovis, 2sinin ise B. bovis + B. bigemina ile miks enfekte olduğu saptanmıştır. Sonuç olarak bu çalışma ile sığırlarda B. bovis ve B. bigeminanın araştırılmasında Real Time PCR yönteminin Nested PCR ve RLB tekniklerine oranla daha duyarlı olduğu belirlenmiştir.

Comparative diagnosis of Babesia bovis and Babesia bigemina in Cattle by Reverse Line Blotting, Nested PCR and real time PCR techniques

This study was carried out to compare Reverse Line Blotting (RLB), Nested PCR and Real Time PCR techniques in the molecular diagnosis of Babesia bovis and Babesia bigemina in cattle. Genomic DNA extractions were performed on 400 blood samples which were previously collected from cattle in various provinces of Turkey and stored in the laboratory with respect to use in different project studies. The concentrations of the DNAs were measured in NanoDrop spectrophotometer and analyzed by RLB, Nested PCR and Real Time PCR techniques after preparing the suitable concentrations. Totally 18 (4.50%), 59 (14.75%), 16 (4.00%) and 2 (0.50%) of examined samples were found to be infected with B. bovis, B. bigemina, Babesia spp. and B. bovis + B. bigemina mix, respectively by RLB. 23 (5.75%), 71 (17.75%), 7 (1.75%) and 23 (5.75%), 75 (18.75%), 9 (2.00%) of the examined samples were found to be infected with B. bovis, B. bigemina and B. bovis + B. bigemina mix by Nested PCR and Real Time PCR, respectively. When comparing the Nested PCR and RLB results with Real Time PCR assay, 94.4% and 88.8% sensitivity and both 100.0% specificity were determined, respectively. 5, 9 and 2 out of the total 16 Babesia spp. positivitys in RLB test were determined as B. bigemina, B. bovis, B. bovis + B. bigemina mix, respectively by both Real Time and Nested PCR. In conclusion, Real Time PCR was found to be more sensitive than Nested PCR and RLB in the investigation of B. bovis and B. bigemina in cattle with this study.

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