NESTED PCR ASSAY FOR DETECTION OF GRANULOCYTIC EHRLICHIAE

A sensitive and specific nested PCR assay was developed for the detect ion of granulocytic ehrlichiae, The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtai ned from seven humans with clinical presentations compatible with huma n granulocytic ehrlichiosis, Five of the seven suspected cases were po sitive by the PCR assay using DNA extracted from whole blood as the te mplate, compared with a serologic assay that identified only one posit ive sample, The PCR assay using DNA extracted from the corresponding s erum samples as the template identified three positive samples. The se nsitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene, T he application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks col lected in Rhode Island and from EDTA-blood specimens obtained from whi te-tailed deer in Maryland. All PCR products were sequenced and identi fied as specific to granulocytic ehrlichiae. A putative variant granul ocytic ehrlichia 16S rRNA gene sequence was detected among products am plified from both the ticks and the deer blood specimens.