Submitted to: American Society of Microbiologists Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 3/7/2005
Publication Date: 6/8/2005
Citation: Yeh, H., Shoemaker, C.A., Klesius, P.H. 2005. Rapid, specific, and sensitive detection of flavobacterium columnare by a loop-mediated isothermal amplification method. American Society of Microbiologists Abstracts.
Technical Abstract: Background: Columnaris disease caused by Flavobacterium columnare is an economically important disease in aquaculture worldwide. Early rapid diagnosis of the pathogen is one of means for effective control the disease. A loop-mediated isothermal amplification assay that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated and optimized for Flavobacterium columnare detection. Methods: A set of four primers, two outer and two inner, was designed specifically to recognize the 16S ribosomal DNA gene of this pathogen. Bacterial DNA was prepared by lysis of the bacterial pellet in a lysis buffer and boiled for 10 min. The LAMP reaction was carried out in a 25-'l reaction mixture containing (final concentrations): 1X reaction mix with 8 mM MgSO4, 0.8 M betaine, 1.0 mM dNTP, 0.2 'M each of F3 and B3 primers, 1.6 'M each of FIP and BIP, 0.32 units/'l Bst DNA polymerase and appropriate amount of template genomic DNA. The reaction was carried out at 65 oC for one hr and inactivated at 80 oC for 10 min (Notomi et al. 2000). The amplified products (3 'l/well) were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. Results: Our results show that the ladder-like pattern of bands sizes from 195 bp specifically to the Flavobacterium columnare 16S ribosomal DNA gene was amplified. The detection limit of this LAMP assay was about 20 colony forming units. In addition, this optimized LAMP assay was used to detect the Flavobacterium columnare 16S ribosomal DNA gene in experimentally challenged channel catfish. Conclusion: We concluded that the LAMP assay can potentially be used for rapid diagnosis of Flavobacterium columnare in hatcheries and ponds.