Submitted to: Aquaculture America Conference
Publication Type: Abstract Only
Publication Acceptance Date: October 7, 2004
Publication Date: January 17, 2005
Citation: Yeh, H., Shoemaker, C.A., Klesius, P.H. 2005. Use of loop-mediated isothermal amplification for rapid detection of channel catfish ictalurus punctatus important bacterial pathogen. Aquaculture America Conference. Technical Abstract: Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in $40 - 50 million annual losses in profits to catfish producers. Early detection of this pathogen will be necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal amplification method (LAMP) developed by Notomi et al. (2000) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of this channel catfish important pathogen. Sets (309 and 919) of four primers, two outer and two inner, were designed specifically to recognize EIP gene for Edwardsiella ictaluri. Bacterial genomic DNA templates were prepared by hot lysis of cultured cell suspension in a lysis buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, pH 8.0 and 1.2% Triton X-100). Amplification of the specific gene segments were carried out at 65 oC. The amplified gene products were analyzed by agarose gel electrophoresis and detected by staining with ethidium bromide. Our results show that the 309 set specifically amplified Edwardsiella ictaluri gene, but not the 919 set, within 60 min, indicating that the LAMP method can potentially be used for rapid diagnosis in the field.