SPECIFIC DETECTION OF COXIELLA-BURNETII THROUGH PARTIAL AMPLIFICATIONOF 23S RDNA

A previously published sequence of the 23S rRNA gene of Coxiella burne tii has been reported to contain an intervening sequence of 444 base p airs (bp). The sequence information on the intervening sequence and th e 23S rRNA gene was exploited to develop a specific PCR-based assay fo r C. burnetii. A primer set was designed that amplified a 477-bp fragm ent encompassing part of the intervening sequence and part of the 23S rDNA. From all of nine C. burnetii strains tested, a fragment of the e xpected size was amplified. As predicted from the published sequence, restriction endonuclease digestion of the PCR product from the Coxiell a strains with RsaI produced two distinct fragments approximately 210- and 270-bp in size. The PCR-based method showed a detection limit of 10(2) bacteria as determined by visualization of the amplicon on an ag arose gel. When experimentally infected blood was analyzed, the detect ion limit was 10(3) bacteria. No visible amplicons were observed when 41 bacterial strains, representing 29 species other than C. burnetii, were tested. The presence of the DNA in all bacterial samples was conf irmed by amplification of a 350-bp fragment of the 16S rDNA using two universal primers. The described method proved to be specific for C. b urnetii and may become a rapid and sensitive diagnostic assay for C. b urnetii. The results also demonstrate that the intervening sequence wi thin the 23S rRNA gene is generally found among isolates of C. burneti i.