DNA PROBES FOR DETECTING COXIELLA-BURNETII STRAINS

Methods have been developed for the rapid detection of C. burnetii by specific hybridization of labelled DNA probes to rickettsial plasmid DNA sequences present in clinical samples. One DNA probe detects all C. burnetii strains, while additional probes differentiate, between organisms associated with chronic or acute disease. Using these probes, C. burnetii can be identified in blood, urine, and tissue samples. The plasmid-derived DNA probes detect as few as 10(4) organisms and less than 1 ng of Coxiella DNA. Host-cell DNA has no effect on the hybridization signal from C. burnetii DNA, and these probes do not cross-react with a variety of microorganisms, including both common laboratory contaminants and organisms that cause clinical symptoms similar to those of Q fever. The sensitivity of the assay is markedly enhanced when the procedure employs the polymerase chain reaction (PCR) to amplify C. burnetii DNA. This requires construction of oligonucleotide primers to DNA sequences flanking the target region of the DNA being amplified. For C. burnetii detection, several sets of primers have been prepared. One set is derived from the QpH1 H fragment, a region that is shared by all C. burnetii plasmids (homologous sequences are also present in the plasmidless strains of C. burnetii). The H primers detect all strains of C. burnetii. To differentiate between C. burnetii strains, additional primers, specific for DNA sequences that are unique either to chronic or acute disease-related strains of C. burnetii are employed. PCR amplifies target sequences up to 10(6)-fold. When DNA hybridization is used in conjunction with PCR, the test can detect less than 10 C. burnetii cells. In addition to providing a useful diagnostic method for detecting C. burnetii in clinical samples, this method is a powerful tool for studying the efficiency of antibiotic therapy, mechanisms of pathogenesis, and/or the nature of persistent infections.