Detection of Rickettsia tsutsugamushi by Gene AmpUcation Using Polymerase Chain Reaction Techniques”a

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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.