دانلود مقاله Detection of Lactococcus garvieae in diseased rainbow trout using specific primer 16S rRNA
سال انتشار: ۱۳۸۷
محل انتشار: اولین کنگره بین المللی مدیریت بهداشتی و بیماریهای آبزیان
تعداد صفحات: ۱
M Soltani – Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
S Haghighi –
A Zargar –
Method & Materials: A number of 72 strains of Gram positive non-motile cocci bacteria were isolated from diseased rainbow trout collected from six provinces. The bacterial isolates were first phenotipically characterized using routine bacteriological works. The bacterial DNA was then extracted from 24 hour culture on nutrient agar supplemented with 5% defibrinated sheep blood at 28◦C. A 16S rRNA PCR amplification was used using forward (CATAACAATGAGAATCGC) and reverse primers (GCACCCTCGCGGGTTG) for identification of L. garviae. These primers amplify a fragment of 1100bp. A 25 μl reaction mixture prepared with 2.5 μl of 10 x PCR buffer, 0.5 μl (10mM) of deoxyribonucletide mixture (dNTP), 1 μl of each primer, 1 μl of purified DNA , 2.5 μl (1.5mM) MgCl2, 0.2μl (1 Unit/μl) of Taq DNA polymerase and sterile deionized distilled water to final volume. The mixture was denatured at 94◦C for 3 min. The amplification of 35 cycles of denaturation at 94◦C for 60 sec, annealing at 53◦C for 60 sec, and extension at 72◦C for 90 sec, ended by an extension step of 10 min at 72◦C.
Results & Conclusion: The results of PCR analysis showed that 36 (50 %) of these bacterial isolates stretch of 1100 bp which exhibit identical size to reference strains of L. garvieae used as the positive control in this study. These strains were isolated from trout farming located in Tehran, Mazandaran, Charmohal va Bakhteyari, Lorestan and Kermansha Provinces during 2007 to 2008.