Semiquantitative species-specific detection of Bartonella henselae and Bartonella quintana by PCR-enzyme immunoassay

Bartonella henselae is the main causative agent of cat-scratch disease, and both B. henselae and Bartonella quintana cause angioproliferative disorder s such as bacillary angiomatosis. To increase the sensitivity of Bartonella detection by PCR and to improve the species differentiation, we developed a semiquantitative, species-specific PCR-based enzyme immunoassay (EIA), Th e 16S rRNA gene was selected as the target sequence. Internal nucleotide se quences derived from the amplified 165 rRNA region were used to develop spe cies-specific oligonucleotide probes for B. henselae and B. quintana. Bioti n-labeled PCR products were immobilized on streptavidin-coated microtiter p lates, hybridized to a digoxigenin-labeled probe, and detected with antidig oxigenin peroxidase conjugate. No cross-hybridization with other Bartonella or non-Bartonella species was observed. This EIA was as sensitive as dot b lot hybridization and was 10 times more sensitive than visualization of PCR products on agarose gels, Serial dilutions of B, henselae and B. quintana suspensions demonstrated that an optical density (OD) of approximately 0.20 0 was equivalent to 5 CFU in the reaction mixture. By comparing the OD of t he bacterial dilutions with that obtained from clinical specimens we could determine that the number of CFU in clinical samples ranged from 10(3) to 1 0(6) CFU/ml, The PCR-EIA developed in the present study is a rapid, sensiti ve, and simple method for the diagnosis of B. henselae and B. quintana infe ctions.