Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/10/2006
Publication Date: 8/8/2006
Citation: Yeh, H., Shoemaker, C.A., Klesius, P.H. 2006. Use of loop-mediated isothermal amplification method for rapid detection of Edwardsiella ictaluri and Flavobacterium columnare in channel catfish. 11th International Symposium on Veterinary Epidemiology and Economics. p.272.
Technical Abstract: After two decades of fast growth, aquaculture will continue to be a crucial food-producing industry worldwide. Infectious diseases are a critical limiting factor in aquaculture. Traditionally, isolation and a series of biochemical tests are required for pathogen identification. However, these procedures that may take at least two days are not practical in the field. PCR has recently been reported for rapid detection of fish pathogens, but Taq DNA polymerase in PCR assays is often inactivated by inhibitors present in crude biological samples. Conventional PCR requires specialized, high-cost instruments and consumables. Thus, a rapid, simple and cost-effective assay alternative to PCR is needed. In this communication, the loop-mediated isothermal amplification method (LAMP) based on autocycling strand displacement DNA synthesis in the presence of Bst DNA polymerase under isothermal condition within one hour (Notomi et al., Nucleic Acids Res. 15, e63, 2000) was evaluated and optimized for rapid diagnosis of two important channel catfish pathogens. Sets of four primers were designed specifically to recognize the eip 18 and 16S rDNA genes of Edwardsiella ictaluri and Flavobacterium columnare, respectively. The LAMP reaction was performed at 65 oC for one hr. Our results show that the ladder-like pattern of specific DNA bands was amplified. The detection limit was about 76 fg and 230 fg of E. ictaluri and F. columnare genomic DNA, respectively. Also, this optimized assay could detect the eip 18 and 16S rDNA genes in respective E. ictaluri and F. columnare infected channel catfish. We concluded that the LAMP assay is useful for rapid detection and epidemiological studies of these Gram-negative pathogens.