Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/3/2005
Publication Date: 4/8/2006
Citation: Yeh, H., Shoemaker, C.A., Klesius, P.H. 2006. Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method. Journal of Applied Microbiology, 100 (2006) 919-925. Interpretive Summary: Channel catfish production is the largest aquacultural industry in the southeastern United States. Its annual output reaches 410 million dollars, but often succumbs to Flavobacterium columnare infection resulting in hundreds of thousands of dollars annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of F. columnare. A set of four primers, two outer and two inner, was designed specifically to recognize the 16S ribosomal RNA gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the 16S ribosomal RNA gene were also optimized at 65 oC for 60 minutes, respectively. Our results show that ladder-like band patterns sizes from 204 bp specifically to the F. columnare gene was amplified. The detection limit of this LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was used to detect the F. columnare 16S ribosomal RNA gene in brains of experimentally infected channel catfish. Thus, we concluded that the LAMP assay can be potentially used for rapid diagnosis in hatcheries and ponds.
Technical Abstract: Aims: To evaluate the loop-mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus. Methods and Results: A set of four primers, two outer and two inner, were designed specifically to recognize 16S ribosomal RNA gene of this pathogen. Bacterial genomic DNA templates were prepared by hot lysis in a lysis buffer. Amplification of the specific gene segments were carried out at 65 oC for one hour. The amplified gene products were analyzed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. Our results demonstrate that the ladder-like pattern of bands from 204 bp specific to the Fl. columnare 16S ribosomal RNA gene was amplified. The detection limit of the LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was able to detect the Fl. columnare 16S ribosomal RNA gene in experimentally infected channel catfish. Conclusions: The LAMP assay for Fl. columnare detection in channel catfish was established. Significance and Impact of the Study: Because LAMP assay is a rapid, sensitive, specific, simple and cost-effective assay for Fl. columnare detection in channel catfish, it is useful for rapid diagnosis of Fl. columnare in fish hatcheries and the field.