Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes

TLDR

The authors amplified citrate synthase and a large antigen gene using PCR with specific primers, then digested the products with restriction enzymes to generate fragment patterns that differentiate rickettsial species and estimate intraspecies sequence divergence. This approach overcomes previous technical obstacles and yields genotypic relationships that align well with established rickettsial taxonomy.

Abstract

DNA sequences from specific genes, amplified by the polymerase chain reaction technique, were used as substrata for nonisotopic restriction endonuclease fragment length polymorphism differentiation of rickettsial species and genotypes. The products amplified using a single pair of oligonucleotide primers (derived from a rickettsial citrate synthase gene sequence) and cleaved with restriction endonucleases were used to differentiate almost all recognized species of rickettsiae. A second set of primers was used for differentiation of all recognized species of closely related spotted fever group rickettsiae. The procedure circumvents many technical obstacles previously associated with identification of rickettsial species. Multiple amplified DNA digest patterns were used to estimate the intraspecies nucleotide sequence divergence for the genes coding for rickettsial citrate synthase and a large antigen-coding gene of the spotted fever group rickettsiae. The estimated relationships deduced from these genotypic data correlate reasonably well with established rickettsial taxonomic schemes.