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

Abstract

DNA sequences from specific genes, amplified by the polymerase chain reaction technique, were used as substrata for nonisotopic restriction endonuclease fragment klgth polymorphism differentiation of rickettsial species and genotypes. The products amplified using a single pai 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 al 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 estimted relationships deduced from these genotypic data correlate reasonably well with established rickettsial taxonomic schemes.

First identification of natural infection of Rickettsia rickettsii in the Rhipicephalus sanguineus tick, in the State of Rio de Janeiro

“…Total DNA was extracted from the pulverized ticks using QIAamp DNA Mini Kit (QIAGEN TM ). DNA was divided in pools organized by locality, species and sex and were screened for the presence of Rickettsia DNA by polymerase chain reaction (PCR) using four set of primers: Rr190.70p/ Rr190.602n (OmpA) (Regnery et al 1991), BG1-21/BG2-20 (OmpB) (Eremeeva et al 1994), Tz15/Tz16 (17 kDa protein enconding-gene.) (Tzianabos et al 1989) and RpCS.877p/ RpCS.1258n (gltA) (Roux et al 1997).…”

Importance of Common Wall Lizards in the Transmission Dynamics of Tick-Borne Pathogens in the Northern Apennine Mountains, Italy

“…Detection of Rickettsia spp. in ticks was performed by targeting the citrate synthase (gltA) [22], OmpA [41] and OmpB genes [46]. Rickettsia spp.…”

Spotted fever group Rickettsia infecting ticks (Acari: Ixodidae) in the state of Santa Catarina, Brazil

“…In each set of reactions, negative control tubes containing water and a positive control tube containing DNA of the strain NOD of Rickettsia parkeri were included (Ogrzewalska et al 2009). Samples that yielded visible amplicons of the expected size by the gltA-PCR were further tested by a second PCR assay using primers Rr190.70p and Rr190.602n targeting a 532-bp fragment of the rickettsial gene ompA, as previously described (Regnery et al 1991). All ompA-PCR amplicons of the expected size were submitted to direct DNA sequencing using an automated ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).…”