Aims: Loop‐mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a protocol for detecting Nocardia seriolae which is a causative agent of fish nocardiosis, was designed.
Methods and Results: A set of four primers, two inner and two outer, were designed based on the sequence of the 16S–23S ribosomal RNA internal transcribed spacer region of N. seriolae. Time and temperature conditions for detection of N. seriolae were optimized for 60 min at 65°C. Other fish pathogen was not amplified by this LAMP system. The detection of N. seriola using LAMP was found to be more sensitive than that by polymerase chain reaction.
Conclusions: LAMP is a highly sensitive and rapid diagnostic procedure for detection of N. seriolae.
Significance and Impact of the Study: LAMP is a useful diagnostic method for fish nocardiosis.
Challenge methods for inducing nocardiosis, caused by Nocardia seriolae in yellowtail, Seriola quinqueradiata, were evaluated. The first method involved intraperitoneal injection of 1.1 x 10(2), x 10(3) and x 10(4) cfu of N. seriolae; the second was by bath immersion with three different concentrations of bacterial suspension for 10 min; the third was by intradermal injection using a multipuncture device; the fourth was by oral administration using a tube; the fifth was based on the co-habitation of uninfected fish with others that had been artificially infected, i.e. intraperitoneally or by immersion for 10 min. The LD(50) values for the infection trials were 1.9 x 10(2) (intraperitoneal injection), 1.5 x 10(4) mL(-1) (immersion), 4.3 x 10(6) (intradermal injection) and 1.7 x 10(7) (oral administration). In the co-habitation challenge, mortalities were 70% and 50% in groups of non-infected fish mixed with fish infected by the i.p. injection and immersion methods, respectively. Fish challenged by intraperitoneal injection developed adhesions. Fish challenged by other methods did not show any gross clinical signs and moribund fish were similar to natural infection.
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