PCR and sequencing of independent genetic targets for the diagnosis of culture negative bacterial endocarditis

Molecular methods utilizing broad-range primers for 16S rDNA PCR and sequen cing have been widely evaluated for their utility in culture negative diagn ostic bacteriology. Difficulties in determining the incidence of false posi tive PCR results, especially in the absence of an equally sensitive confirm atory method however, have prevented wide clinical use of this sensitive te chnology. Here we report two cases of culture-negative endocarditis, in whi ch PCR using 16S rDNA broad-range primers generated sequences specific for Bartonella spp. and Streptococcus oralis, respectively. To confirm these re sults, a second species- or genus-specific molecular target was chosen for each organism and detected in the split samples sequencially. Thus, molecul ar detection of a second species-specific target can be used to confirm PCR results generated from 16S rDNA broad-range primers and to control for pot ential false positive results due to environmental and amplicon carry-over contamination during specimen processing and testing in the laboratory. (C) 2001 Elsevier Science Inc. All rights reserved.