Nikolina RUSENOVA, Mihail CHERVENKOV, Ivo SIRAKOV

Enzootik Sığır Lökozunun Rutin Teşhisi İçin Kullanılan Dört Laboratuvar Testinin Karşılaştırılması

Bu çalışmanın amacı, agar jel immünodifüzyon testi (AGID) ve iki tip PCR’nin, nested PCR (kandan nükleik asit ekstraksiyonu ile birlikte) ve kanda direkt nested PCR (db nested PCR) - ekstraksiyon olmaksızın, tanısal yeteneklerinin bu hastalığın rutin teşhisinde altın standart olarak kullanılan Enzyme-Linked Immunosorbent Assay (ELISA) ile karşılaştırılmasıydı. Çalışmada, 18 ay ile 5 yaş arası sığırlardan toplam 409 kan örneği toplandı ve tüm örnekler bu dört yöntem ile analiz edildi. İlk analizleri takiben, örnekler -20°C’de saklandı ve sonuçların tekrarlanabilirliği açısından bir aylık dondurularak depolamanın ardından dört yöntem ile yeniden analiz edildi. ELISA ile 57 (%13.9) hayvan pozitif saptanırken, AGID ile 33 ve her iki tip nested PCR ile 56 hayvan pozitif saptandı. ELISA’ya kıyasla AGID, %57.9’luk düşük sensitivite ve orta düzeyde uyum gösterdi. Ayrıca AGID, iki bağımsız ölçümden elde edilen sonuçlarla da tutarlılık göstermedi. İki tip nested PCR yöntemi de 0.99 kappa değeriyle ELISA ile neredeyse tam uyum gösterdi. Bu çalışmada kullanılan diğer tekniklere kıyasla AGID, 22 örnekte daha düşük sensitivite gösterdiği için ve tekrarlanabilirliği olmadığı için kan örneklerinde Enzootik sığır lökozunun teşhisinde bu testin gelecekte uygulanmasının yeniden düşünülmesi gerektiğini öneriyoruz. Diğer taraftan, db nested PCR çok iyi bir sensitivite ve tekrarlanabilirlik gösterdi, ayrıca daha az örnek işlenmesine ihtiyaç duydu. Bütün bunlar, db nested PCR’yi rutin teşhis için potansiyel olarak uygun hale getirmektedir

Comparison Between Four Laboratory Tests for Routine Diagnosis of Enzootic Bovine Leukosis

The aim of this study was to compare the diagnostic capabilities of the agar gel immunodiff usion test (AGID) and two types of PCR, nested PCR (with nucleic acid extraction from blood) and direct blood nested PCR (db nested PCR) - without extraction, vs. enzyme-linked immunosorbent assay (ELISA) as the gold standard in the routine diagnostics of this disease. A total of 409 blood samples were obtained from cattle 18 mo. to 5 yrs. of age, and all the samples were analyzed using the four assays. Following the initial testing, the samples were stored at -20°С and re-tested using all the four techniques after a month of freeze storage to determine the reproducibility of the results. ELISA detected 57 animals as positive (13.9%) versus 33 ones using AGID and 56 using the two types of nested PCR. AGID showed low sensitivity of 57.9% and moderate agreement compared to ELISA. In addition, AGID did not show consistency in the results from the two independent measurements. The two types of nested PCR showed nearly full agreement with ELISA with a kappa value of 0.99. Since AGID showed lower sensitivity and lack of reproducibility in the results for 22 samples as compared to the other techniques used in this study, we suggest that the future application of this test for the diagnosis of Enzootic bovine leucosis in blood samples should be reconsidered. On the other hand, db nested PCR demonstrated very good sensitivity and reproducibility of results, it also requires less sample processing. All this makes it potentially suitable for routine diagnostics.

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