Development of polymerase chain reaction assays for detection, identification, and differentiation of Piscirickettsia salmonis

A nested polymerase chain reaction (PCR) was developed to detect genomic DNA of Pisci-rickettsia salmonis, the causative agent of an epizootic disease in salmonids. The nested PCR assay, which used general bacterial 16S rDNA primers in the first amplification reaction, and Fl salmonis-specific primers in a second reaction, allowed detection of less than 1 P. salmonis tissue culture infectious dose 50 (TCID50). Using the Fl salmonis-specific primers in a single PCR reaction allowed the detection of 60 TCID50. The specificity of the PCR was assessed with a panel of 1: salmonid and 15 bacterial genomic DNA preparations. Amplification products were produced only with I? salmonis DNA. Restriction fragment length polymorphism (RFLP) analysis of the complete 16S gene PCR products demonstrated that 1 isolate, EM-90, was unique. Two additional primers were developed and used in PCR assays that differentiated EM-90 from the 4 other P. salmonis isolates tested.