Development and Validation of an Improved PCR Method Using 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals.

Kakumanu ML1, Ponnusamy L1, Sutton HT1, Meshnick SR2, Nicholson WL3, Apperson CS4.

Author information

Abstract

A novel nested PCR assay was developed to detect Rickettsia spp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer of Rickettsia spp. The newly designed primers were evaluated using genomic DNA from 11 Rickettsia species, belonging to the spotted fever, typhus, and ancestral groups and in parallel compared to other Rickettsia-specific PCR targets (ompA, gltA and 17-kDa). The new 23S-5S nested PCR assay amplified all 11 Rickettsia spp. but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from "Candidatus R. amblyommii". Subsequently, the detection efficiency of the 23S-5S nested assay was compared to the other three assays using genomic DNA extracted from 40 adult Dermacentor variabilis ticks. The nested 23S-5S assay detected Rickettsia DNA in 45% of the ticks while the amplification rate of the three other assays ranged between 5-20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species of Rickettsia. The nested 23S-5S PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species of Rickettsia in the ticks. Rickettsia amblyommii, R. montanensis, R. felis and R. bellii were frequently identified species along with some potentially novel Rickettsia that were closely related to R. bellii and R. conorii.