Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.
Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in 40--50 million 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 E. ictaluri. A set of four primers, two outer and two inner, was designed specifically to recognize the eip 18 gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the eip 18 gene were also optimized at 65 degrees C for 60 min, respectively. Our results show that the ladder-like pattern of bands sizes from 234 bp specifically to the E. ictaluri 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 E. ictaluri eip 18 gene in brains of experimentally challenged channel catfish. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in hatcheries and ponds.
Yeh
Hung-Yueh
HY
United States Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research Unit, Auburn, AL 36831-0952, USA. yyeh@ars.usda.gov
Shoemaker
Craig A
CA
Klesius
Phillip H
PH
D000818:Animals; D001921:Brain / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics; D020610:Edwardsiella ictaluri / Q000235:genetics / Q000302:isolation & purification*; D004756:Enterobacteriaceae Infections / Q000382:microbiology / Q000662:veterinary; D005393:Fish Diseases / Q000382:microbiology; D005798:Genes, Bacterial / Q000235:genetics; D007059:Ictaluridae / Q000382:microbiology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers