Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA

Abstract

Currently, the genotypic identification of the spotted fever group (SFG) rickettsiae is based on restriction fragment length polymorphism analysis of PCR-amplified genes coding for the enzyme citrate synthase and the surface proteins rOmpA and rOmpB. A set of useful restriction endonucleases was found following comparison of Rickettsia rickettsii and R. prowazekii sequences. However, by using three PCR amplifications and four enzyme digestions with this set, it was impossible to differentiate between all of the known serotypes of the SFG rickettsiae. We amplified by PCR and sequenced using an automated laser fluorescent DNA sequencer a fragment of the gene encoding the protein rOmpA from 21 serotypes of the SFG rickettsiae. A 632-bp amplification product was obtained for most of the strains, although no product could be obtained by using R. akari, R. australis, R. helvetica, and R. bellii DNAs. We found a characteristic sequence for all strains studied except the two isolates of R. massiliae, isolates GS and Mtul. Using the software package BISANCE, we determined the restriction map of this fragment and identified five potentially useful endonucleases, RsaI, AluI, PstI, XbaI, and AvaII. We confirmed the computer analysis-derived profiles by PCR-restriction fragment length polymorphism analysis. The combination of the profiles obtained after digestion of the PCR product by RsaI and PstI allowed for the differentiation of 16 strains. The use of AluI and XbaI allowed for the characterization of R. parkeri and strain HA-91, respectively. R. africae and strain S were differentiated by AvaII digestion. Thus, using a single PCR amplification, we were able to differentiate all of the SFG rickettsiae whose ompA gene was amplified by PCR.