Gıda patojenlerinin tespiti için floresan temelli ekonomik DNA test yöntemlerinin geliştirilmesi
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Tarih
2015
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Çanakkale Onsekiz Mart Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Gıdalarda mikrobiyal patojen tespiti için real-time PCR tekniklerinin geniş uygulama alanı bulması, ayrıca güvenilir, hızlı, kolay ve ekonomik metotlara artan endüstriyel ilgi, DNA temelli metotların geliştirilmesine yol açmıştır. Bu tezde, Listeria monocytogenes ve Escherichia coli O157:H7'nin kompleks bir gıda matrisinde pratik ve eş zamanlı tespiti için ekonomik bir PCR tekniğinin geliştirilmesi anlatılmıştır. Bakteriyel DNA Multi-Fast DNA İzolasyon Kiti (GIDAGEN, Türkiye) ile izole edilmiştir. GC içeriği birbirinden oldukça farklı olan yayınlanmış primerlerin kombinasyonu ve değerlendirilmesi ile EvaGreen temelli dubleks real-time PCR sistemi geliştirilmiştir. Öncül dubleks PCR, model gıda sistemlerinde L. monocytogenes ve E. coli O157:H7'ye uygulanmıştır. L. monocytogenes ve E. coli O157:H7 tespiti için sırasıyla hylA ve uidA hedef gen bölgeleri çoğaltılmıştır. EvaGreen temelli simpleks ve dubleks real-time PCR sistemi ile spesifik PCR ürünleri erime eğrisi analizleri ile belirlenmiş ve L. monocytogenes için tekrarlanabilir Tm değeri 77.00±0.4°C iken, E. coli O157:H7'nin tekrarlanabilir Tm değerinin 85.60±0.5°C olduğu gösterilmiştir. Primerlerin spesifitesi 10 tane farklı bakteri suşu kullanılarak test edilmiştir ve L. monocytogenes ve E. coli O157:H7 tespiti için primerlerin spesifik olduğu belirlenmiştir. EvaGreen temelli dubleks ve simpleks real-time PCR için mutlak algılama sınırı iki patojen bakteri DNA'sı için de 0.00001 ng/?l olarak bulunmuştur. Simpleks ve dubleks tekniğin göreceli tesbit limiti her iki patojen için 10 hücre/ml'den düşük bulunmuştur.
The wide application of real-time PCR techniques and the increasing industrial interest towards rapid, easy, economical and reliable methods have led to the development of DNA based methods for the detection of microbial pathogens in food. In the present paper, we describe the development of a cost-efficient PCR technique for simultaneous and practical detection of Listeria monocytogenes and Escherichia coli O157:H7 in a complex food matrix. Bacterial DNA was isolated with Multi-Fast DNA Isolation Kit (GIDAGEN, İstanbul, Turkey). An EvaGreen based duplex PCR system was developed with the evaluation and combination of published primers whose GC contents were quite different from one another. Novel duplex PCR was applied to L. monocytogenes and E. coli O157:H7 in model food systems. Target genes hylA and uidA were amplified for the detection of L. monocytogenes and E.coli O157:H7, respectively. EvaGreen based simplex and duplex real-time PCR showed that specific PCR products were identified by melting curve analysis, and a reproducible Tm of 77.00± 0.4°C for L. monocytogenes and 85.60±0.5 for E. coli O157:H7. The specifity of primers were evaluated in 10 different bacterial strains and showed that the chosen primers were specific for the detection of L. monocytogenes and E. coli O157:H7 by real-time PCR. The absolute detection limit of EvaGreen based simplex and duplex real-time PCR was 0.00001 ng/?l for DNAs for both pathogens. The relative detection limit of simplex and duplex technique was less than 10 cells/ml for two pathogens.
The wide application of real-time PCR techniques and the increasing industrial interest towards rapid, easy, economical and reliable methods have led to the development of DNA based methods for the detection of microbial pathogens in food. In the present paper, we describe the development of a cost-efficient PCR technique for simultaneous and practical detection of Listeria monocytogenes and Escherichia coli O157:H7 in a complex food matrix. Bacterial DNA was isolated with Multi-Fast DNA Isolation Kit (GIDAGEN, İstanbul, Turkey). An EvaGreen based duplex PCR system was developed with the evaluation and combination of published primers whose GC contents were quite different from one another. Novel duplex PCR was applied to L. monocytogenes and E. coli O157:H7 in model food systems. Target genes hylA and uidA were amplified for the detection of L. monocytogenes and E.coli O157:H7, respectively. EvaGreen based simplex and duplex real-time PCR showed that specific PCR products were identified by melting curve analysis, and a reproducible Tm of 77.00± 0.4°C for L. monocytogenes and 85.60±0.5 for E. coli O157:H7. The specifity of primers were evaluated in 10 different bacterial strains and showed that the chosen primers were specific for the detection of L. monocytogenes and E. coli O157:H7 by real-time PCR. The absolute detection limit of EvaGreen based simplex and duplex real-time PCR was 0.00001 ng/?l for DNAs for both pathogens. The relative detection limit of simplex and duplex technique was less than 10 cells/ml for two pathogens.
Açıklama
Fen Bilimleri Enstitüsü, Biyomühendislik ve Malzeme Mühendisliği Ana Bilim Dalı
Anahtar Kelimeler
Biyomühendislik, Bioengineering, Genetik
