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Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop‐mediated isothermal amplification method
Author(s) -
Yeh H.Y.,
Shoemaker C.A.,
Klesius P.H.
Publication year - 2006
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2006.02853.x
Subject(s) - ictalurus , loop mediated isothermal amplification , catfish , ethidium bromide , microbiology and biotechnology , biology , genomic dna , 16s ribosomal rna , agarose gel electrophoresis , polymerase chain reaction , ribosomal rna , gene , dna , genetics , fishery , fish <actinopterygii>
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 was carried out at 65°C for 1 h. The amplified gene products were analysed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. A PCR assay was also included in this study. 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.