Specific amplification of Ehrlichia platys DNA from blood specimens by two-step PCR

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Abstract
A two-step PCR method for diagnosis of canine infectious cyclic thrombocytopenia was established. Three primers derived from the 16S and rRNA gene sequence were used to amplify genomic DNA specifically from Ehrlichia platys. Two-round amplification with DNA templates prepared from E. platys-infected blood specimens produced 742- and 385-bp fragments, but these products were not found when an Ehrlichia canis-infected blood sample and Escherichia coli were used. This method, for which the minimum detectable copy number in the blood specimen was estimated to be five ehrlichia inclusion within platelets, is more sensitive than single PCR amplification. These results demonstrate that this two-step PCR is highly sensitive and efficient for detecting the etiologic agent of canine infectious cyclic thrombocytopenia in blood. The same technique was applied to blood specimens collected from a dog inoculated with E. platys. Amplification of the target DNA fragments was observed with blood collected on the fifth day after inoculation, which indicates that this method is also feasible for early diagnosis of E. platys infection.