Balarılarındaki Amerikan yavru çürüklüğü hastalığı etkeni olan Paenibacillus larvae'nın larvalı petek ve bal örneklerinden konvansiyonel ve moleküler biyolojik yöntemlerle teşhisi
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Dosyalar
Tarih
2017
Yazarlar
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Ülkemizde AYÇ hastalığı teşhisi, Gıda, Tarım ve Hayvancılık Bakanlığı'nın "Laboratuarlar Arası Metot Birliği" kitapçığına göre, konvansiyonel kültür yöntemi ile yapılmaktadır. Konvansiyonel kültür yöntemi ile teşhis, uzun sürmekte ve maliyeti yüksek olmaktadır. Bazı ülke ve laboratuarlarda OIE tarafından da tavsiye edilen moleküler yöntemler kullanılarak daha hızlı sonuçlar alınabilmektedir. Bu çalışma ise, teşhis prosedüründe uygulanabilirliği açısından konvansiyonel kültür yöntemi, Konvansiyonel PCR ve Real-Time PCR yöntemlerinin paralel olarak uygulanacağı bir metot karşılaştırma projesi olarak tasarlanmıştır. Bu amaçla farklı kaynaklardan 96 adet larvalı petek, ballı petek ile az sayıda kabartılmış petek ve ergin arı örnekleri toplanmıştır. Örnekler önce konvansiyonel kültür metodu ile incelenmiş, daha sonra örneklerden DNA izolasyonu yapılarak Konvansiyonel PCR ve Real-Time PCR metotları ile incelenmiştir. Moleküler çalışmalar için, literatürlerde belirtilen primer çiftleriyle sonuç alınamamış, üç başarısız denemeden sonra ancak dördüncü çalışmada PCR için farklı, Real-Time PCR için farklı primerler kullanılarak metotlar optimize edilebilmiştir. Sonuç olarak, konvansiyonel kültür metodu ile 10, konvansiyonel PCR ile 16, Real-Time PCR ile 25 örnek pozitif olarak bulunmuştur. Kültür yöntemi ile pozitif bulunan tüm örnekler, moleküler yöntemlerle de pozitif bulunmuştur. Çalışmamız sonucundaki bulgular ışığında, AYÇ şüpheli bir örnek, moleküler yöntemlerle muayene edilip negatif bulunursa menfi olarak değerlendirilebilir. Moleküler yöntemlerle pozitif bulunursa, kültür yöntemi ile muayeneye alınıp, konvansiyonel kültür yöntemi sonuçlarına göre değerlendirilmesinin uygun olacağı belirlenmiştir.
In Turkey, diagnosis of American Foulbrood Disease (AFB) is carried out with conventional bacteriological methods according to the "Laboratory Method Unification Manual" of Ministry of Food, Agriculture and Livestock. Diagnosis with conventional culture method is time consuming and expensive. In some countries and laboratories, molecular methods recommended by OIE could be used for faster results. This research was designed as a method comparison work where conventional methods, PCR and Real Time PCR methods will be applied parallel regarding to application on the diagnosis process. For this purpose, 96 samples consisting of larval broods, honey broods and phenomenal amounts of harvested broods and adult bees were taken from different sources. Samples were initially examined by culture methods, and then by conventional PCR and real time PCR methods. In molecular studies optimization was achieved with different primers for conventional PCR and real time PCR methods. As a result, 10 samples were found to be positive with conventional culture methods, 16 were positive with PCR and 25 were positive with Real-Time PCR. All samples found positive with culture method were also found to be positive with molecular methods. According to the findings of our work, an AFB suspicious sample can be considered as negative if it is also was found negative with molecular methods. If it is found to be positive with molecular methods, it would be appropriate that the sample should be examined and results should be evaluated with culture methods.
In Turkey, diagnosis of American Foulbrood Disease (AFB) is carried out with conventional bacteriological methods according to the "Laboratory Method Unification Manual" of Ministry of Food, Agriculture and Livestock. Diagnosis with conventional culture method is time consuming and expensive. In some countries and laboratories, molecular methods recommended by OIE could be used for faster results. This research was designed as a method comparison work where conventional methods, PCR and Real Time PCR methods will be applied parallel regarding to application on the diagnosis process. For this purpose, 96 samples consisting of larval broods, honey broods and phenomenal amounts of harvested broods and adult bees were taken from different sources. Samples were initially examined by culture methods, and then by conventional PCR and real time PCR methods. In molecular studies optimization was achieved with different primers for conventional PCR and real time PCR methods. As a result, 10 samples were found to be positive with conventional culture methods, 16 were positive with PCR and 25 were positive with Real-Time PCR. All samples found positive with culture method were also found to be positive with molecular methods. According to the findings of our work, an AFB suspicious sample can be considered as negative if it is also was found negative with molecular methods. If it is found to be positive with molecular methods, it would be appropriate that the sample should be examined and results should be evaluated with culture methods.
Açıklama
Anahtar Kelimeler
Balarısı, Amerikan Yavru Çürüklüğü, Teşhis, PCR, Real-Time PCR, Honeybee, American Foulbrood, Diagnosis, PCR, Real-Time PCR
