title: Detection of Rickettsia tsutsugamushi by gene amplification using polymerase chain reaction techniques.
abstract: Scrub typhus is commonly undiagnosed in endemic areas due, in part, to dependence on retrospective serodiagnosis. Since the etiologic agent, R. tsutsugamushi, will not grow in cell-free systems, a rapid direct-agent detection system such as provided by polymerase chain reaction (PCR) methodology is needed. Genes coding for the variable 56-kDa antigen of R. tsutsugamushi were amplified through 35 cycles using 20-mer oligonucleotide primers and Taq polymerase. Amplification of 1-ng samples of DNA extracted from purified prototype R. tsutsugamushi Karp, Gilliam, and Kato strains was detected by direct visual inspection of the electrophoresed, ethidium bromide-stained, specific bands. Specificity of the PCR was shown when PCR amplification of various non-scrub typhus rickettsial DNAs was unsuccessful. R. tsutsugamushi DNA extracted from the blood of infected mice could be PCR amplified and the 1477-base pair product detected by either direct visualization or by specific hybridization with amplified non-radioactive digoxigenin-11-dUTP-labeled Karp 56-kDa DNA probe.
authors.affiliation: Rickettsial Diseases Division, Naval Medical Research Institute, Bethesda, Maryland 20814-5055.
authors.forename: D J
authors.initials: DJ
authors.lastname: Kelly
authors.forename: D P
authors.initials: DP
authors.lastname: Marana
authors.forename: C K
authors.initials: CK
authors.lastname: Stover
authors.forename: E V
authors.initials: EV
authors.lastname: Oaks
authors.forename: M
authors.initials: M
authors.lastname: Carl
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000032:analysis*; D051379:Mice; D012285:Orientia tsutsugamushi / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial