Mehmet YÜKSEL, Selahattin SERT, Arzu KAVAZ YÜKSEL, Bülent ÇETİN, Mustafa GÜRSES

KOLORİMETRİK LOOP-MEDIATED İZOTERMAL AMPLİFİKASYON METODU İLE LISTERIA MONOCYTOGENES’İN TAVUK ETLERİNDE HIZLI TESPİTİ

Bu çalışmanın amacı tavuk etlerinden (n:50; bütün kanat, göğüs, baget; toplamda N: 150) Listeria monocytogenes’in tespitinde kolorimetrik loop-mediated izotermal amplifikasyon’un (LAMP) performansını değerlendirmektir. Bu amaçla, tavuk etleri 100-104 CFU/25 g (veya bütün tavuk eti) seviyede L. monocytogenes ve rekabetçi mikrobiyota olarak kullanılan 5 diğer bakteri (Salmonella Typhimurium, Campylobacter jejuni, Campylobacter coli, Staphylococcus aureus, Citrobacter freundii) ile inoküle edildi. Ön zenginleştirme sonrası örnekler geleneksel kültürel, gerçek zamanlı PZR ve LAMP kullanılarak analiz edildi. Virulans hlyA gen’in primer setleri (L. monocytogenes-özgü) hidroksinaftol mavisi (HNB) ile görselleştirilmiş LAMP için kullanıldı. Bu hedef gen 65°C 45 dakikada spesifik primerler kullanılarak çoğaltıldı. Üç metot ile gerçekleştirilen analizlerin sonucunda doğal olarak kontamine olmuş 150 örneğin 9’unda (%6) L. monocytogenes varlığı tespit edildi. LAMP, qPCR ve klasik metot doğal ve yapay olarak kontamine olmuş örneklerden L. monocytgones için aynı tespit performansını gösterdi. Bu sonuçlar HNB-LAMP yönteminin, PCR'a alternatif olarak, izotermal koşullar altında L. monocytogenes'e duyarlı, spesifik, basit, hızlı bir tespit tekniği olarak kullanılabileceğini ve L. monocytogenes'in tavuk etlerinden saptanma potansiyeline sahip olduğunu gösterdi.

Anahtar Kelimeler:

Loop-mediated izotermal amplifikasyon (LAMP), Gerçek zamanlı PZR, Listeria monocytogenes, Tavuk etleri, Hızlı

RAPID DETECTION OF LISTERIA MONOCYTOGENES IN CHICKEN MEATS WITH COLORIMETRIC LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) METHOD

The object of this study is to evaluate the performance of the colorimetric loop-mediated isothermal amplification (LAMP) in detecting Listeria monocytogenes from chicken meats (n: 50; whole wing, breast, drumstick; totally N: 150). For this purpose, the chicken meats were artificially contaminated with 100-104 CFU/25 g (or sample) of L. monocytogenes and 5 others bacteria were used as competitive microbiota. After pre-enrichment, the samples were analyzed using conventional cultural, LAMP and real-time PCR methods. Primer sets of virulence gene hlyA were used for the L. monocytogenes-specific visualized LAMP with hydroxynaphthol blue (HNB) dye and amplified the target gene using specific primers at 65 °C for 45 min. As a result of analysis performed by three methods in the naturally contaminated samples, the presence of L. monocytogenes was detected in 9 of 150 samples (6%). The LAMP, qPCR and conventional method showed similar detection performances for L. monocytogenes in the naturally or artifically contaminated samples. These results demonstrated the use of HNB-LAMP method for, as an alternative to PCR, sensitive and specific to L. monocytogenes under isothermal conditions, could use as a simple, rapid detection technique and has the potential for detection of L. monocytogenes from the chicken meats.

Keywords:

Loop-mediated isothermal amplification (LAMP), Real-time PCR, Listeria monocytogenes, Chicken meats, Rapid,

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