BOHV-1 enfeksiyonunun Real-time PCR ve lamp metotlarıyla karşılaştırılmalı teşhisi ve subtiplendirilmesi
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Tarih
2024
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Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü
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info:eu-repo/semantics/openAccess
Özet
Bovine Herpesvirus 1 (BoHV-1) dünya çapında dağılım gösteren, subklinik, hafif veya şiddetli bir hastalık olarak ortaya çıkan önemli bir sığır patojenidir. Solunum, genital, oküler ve ensefalomiyelit formunda geniş ölçüde değişebilen klinik bulgulara sahiptir. Bu çalışmada, Konya YILET Et Entegre Tesislerine kesime getirilen solunum sistemi klinik bulguları gösteren 150 adet sığırdan nazal swap örnekleri alınmıştır. Örneklerin nükleik asit ekstraksiyonu yapıldıktan sonra maliyet, zaman ve pratikliği açısından daha avantajlı olduğu bildirilen LoopMediatedIsothermalAmplification (LAMP) metodu ile Real-Time PCR (PolymeraseChainReaction) metodu karşılaştırılmıştır. Her iki testte de BoHV-1 pozitif çıkan numunelerin, Madin-DarbyBovineKidneycells (MDBK) hücre kültürüne ekimleri yapılmıştır. İzole edilebilen örneklerin moleküler karakterizasyonunun belirlenmesi için konvansiyonel PCR uygulanmıştır. Araştırmada kullanılan nazal swap örneklerinden 8 adedi (%5,33) Real-Time PCR testi ile pozitif olarak belirlenirken, 13 adedi (%8,66) LAMP testi ile pozitif olarak belirlenmiştir. Yapılan McNemar testi sonucuna göre LAMP ve Real-Time PCR testleri arasında istatistiksel olarak anlamlı bir fark olmadığı (p˃0,05) belirlendi. Hücre kültürüne ekimi yapılan 8 adet örnekten 3'ünde BoHV-1 izole edildi. Bu 3 örneğin PCR işleminden sonra yapılan sekans analizinde, izolatların moleküler karakterizasyonu yapılarak, BoHV-1.1 alt tipe ait olduğu belirlendi.Sonuç olarak BoHV-1 enfeksiyonunun hızlı tanısında LAMP metodunun kullanılabileceği ve enfeksiyonla mücadelede BoHV-1.1 alt tipinin göz önünde bulundurulması gerektiği kanaatine varılmıştır.
Bovine Herpesvirus 1 (BoHV-1) is an important bovine pathogen with a worldwide distribution, presenting as a subclinical, mild or severe disease. It has widely varying clinical manifestations in the form of respiratory, genital, ocular and encephalomyelitis. In this study, nasal swab samples were taken from 150 cattle with respiratory clinical signs brought to Konya YILET Meat Integrated Facility for slaughter. After nucleic acid extraction of the samples, Loop Mediated Isothermal Amplification (LAMP) method, which is reported to be more advantageous in terms of cost, time and practicality, and Real-Time PCR (Polymerase Chain Reaction) method were compared. The samples that were positive for BoHV-1 in both tests were cultured in Madin-Darby Bovine Kidney cells (MDBK) cell culture. Conventional PCR was applied to determine the molecular characterization of the isolated samples. Among the nasal swap samples used in the study, 8 (5.33%) were positive by Real-Time PCR test, while 13 (8.66%) were positive by LAMP test. According to the McNemar test, there was no statistically significant difference between LAMP and Real-Time PCR tests (p˃0,05). BoHV-1 was isolated in 3 of 8 samples cultured in cell culture. In the sequence analysis of these 3 samples after PCR, the isolates were molecularly characterized and determined to belong to BoHV-1.1 subtype. As a result, it was concluded that LAMP method can be used in the rapid diagnosis of BoHV-1 infection and BoHV-1.1 subtype should be considered in the fight against infection.
Bovine Herpesvirus 1 (BoHV-1) is an important bovine pathogen with a worldwide distribution, presenting as a subclinical, mild or severe disease. It has widely varying clinical manifestations in the form of respiratory, genital, ocular and encephalomyelitis. In this study, nasal swab samples were taken from 150 cattle with respiratory clinical signs brought to Konya YILET Meat Integrated Facility for slaughter. After nucleic acid extraction of the samples, Loop Mediated Isothermal Amplification (LAMP) method, which is reported to be more advantageous in terms of cost, time and practicality, and Real-Time PCR (Polymerase Chain Reaction) method were compared. The samples that were positive for BoHV-1 in both tests were cultured in Madin-Darby Bovine Kidney cells (MDBK) cell culture. Conventional PCR was applied to determine the molecular characterization of the isolated samples. Among the nasal swap samples used in the study, 8 (5.33%) were positive by Real-Time PCR test, while 13 (8.66%) were positive by LAMP test. According to the McNemar test, there was no statistically significant difference between LAMP and Real-Time PCR tests (p˃0,05). BoHV-1 was isolated in 3 of 8 samples cultured in cell culture. In the sequence analysis of these 3 samples after PCR, the isolates were molecularly characterized and determined to belong to BoHV-1.1 subtype. As a result, it was concluded that LAMP method can be used in the rapid diagnosis of BoHV-1 infection and BoHV-1.1 subtype should be considered in the fight against infection.
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Aslım, H. P. (2024). BOHV-1 enfeksiyonunun Real-time PCR ve lamp metotlarıyla karşılaştırılmalı teşhisi ve subtiplendirilmesi. (Doktora Tezi). Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, Konya.
