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 Mtu1. 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.

First Molecular Evidence of Zoonotic Bacteria in Ticks in Bosnia and Herzegovina

“…After centrifugation (3000 g/10 s), the supernatant was collected and DNA was extracted with the peqGOLD TriFast Isolation System (Peqlab, Germany) following the manufacturer's instructions. The presence of B. burgdorferi s.l., A. phagocytophilum, 'Candidatus N. mikurensis', SFG rickettsiae and F. tularensis DNA in ticks was tested using PCR protocols published elsewhere (Regnery et al, 1991;Roux et al, 1996;Sj€ ostedt et al, 1997;Liebisch et al, 1998;Brown et al, 2001). Amplified PCR products were visualized by agarose gel electrophoresis, purified and directly sequenced at a commercial company (LGC Genomics, Germany).…”

Fatal Mediterranean spotted fever in Greece

“…Subsequently, the sample was tested for rickettsial IgM and IgG antibodies; no IgM antibodies were detected, whereas a titre of 128 for IgG antibodies against R. conorii was observed. Positive results were obtained with the use of PCRs that amplify a 360-bp fragment of the 17-kDa antigen gene [13], and a 632-bp fragment of the ompA gene [14]. All PCR products were sequenced, allowing postmortem identification of the causative pathogen as R. conorii conorii (the sequences of both genes were identical with the available published sequences from R. conorii strain Malish 7) [6].…”

Life cycle ofRickettsia slovacain L929 cell line studied by quantitative real-time PCR and transmission electron microscopy

“…For Rickettsia sp. detection by PCR, the specific primers RpCS.877p-RpCS.1258n and RR190.70F-RR190.701R were used (Regnery et al, 1991;Roux et al, 1996). Bacterial isolate genome was sequenced by Macrogen Inc. (Seoul, South Korea; http://www.macro gen.com).…”