PCR-RFLP Detection and Genogroup Identification of in Field Samples.

, the causative agent of piscirickettsiosis, is genetically divided into two genomic groups, named after the reference strains as LF-89-like or EM-90-like. Phenotypic differences have been detected between the genogroups, including antibiotic susceptibilities, host specificities and pathogenicity. In this study, we aimed to develop a rapid, sensitive and cost-effective assay for the differentiation of the genogroups. Using an in silico analysis of the 16S rDNA digestion patterns, we have designed a genogroup-specific assay based on PCR-restriction fragment length polymorphism (RFLP). An experimental validation was carried out by comparing the restriction patterns of 13 strains and 57 field samples obtained from the tissues of dead or moribund fish. When the bacterial composition of a set of field samples, for which we detected mixtures of bacterial DNA, was analyzed by a high-throughput sequencing of the 16S rRNA gene amplicons, a diversity of taxa could be identified, including pathogenic and commensal bacteria. Despite the presence of mixtures of bacterial DNA, the characteristic digestion pattern of the genogroups could be detected in the field samples without the need of a microbiological culture and bacterial isolation.