Abstract
A previously published sequence of the 23S rRNA gene of Coxiella burnetii has been reported to contain an intervening sequence of 444 base pairs (bp). The sequence information on the intervening sequence and the 23S rRNA gene was exploited to develop a specific PCR-based assay for C. burnetii. A primer set was designed that amplified a 477-bp fragment encompassing part of the intervening sequence and part of the 23S rDNA. From all of nine C. burnetii strains tested, a fragment of the expected size was amplified. As predicted from the published sequence, restriction endonuclease digestion of the PCR product from the Coxiella strains with RsaI produced two distinct fragments approximately 210- and 270-bp in size. The PCR-based method showed a detection limit of 102 bacteria as determined by visualization of the amplicon on an agarose gel. When experimentally infected blood was analyzed, the detection limit was 103 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 confirmed by amplification of a 350-bp fragment of the 16S rDNA using two universal primers. The described method proved to be specific for C. burnetii and may become a rapid and sensitive diagnostic assay for C. burnetii. The results also demonstrate that the intervening sequence within the 23S rRNA gene is generally found among isolates of C. burnetii.
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References
Marrie TJ. Coxiella burnetii (Q fever). In: Mandell GL, Douglas RGJ, Bennett JE (eds). Principles and practice of infectious diseases, 4th edition. New York: Churchill Livingstone. 1995: 1727–1735.
Stein A, Raoult D. Q fever endocarditis. Eur Heart J 1995; 16(Suppl B): 19–23.
Samuel JE, Frazier ME, Mallavia LP. Correlation of plasmid type and disease caused by Coxiella burnetii. Infect Immun 1985; 49: 775–779.
Stein A, Raoult D. Lack of pathotype specific gene in human Coxiella burnetii isolates. Microb Pathogen 1993; 15: 177–185.
Thiele D, Willems H. Is plasmid based differentiation of Coxiella burnetii in ‘acute’ and ‘chronic’ isolates still valid? Eur J Epidemiol 1994; 10: 427–434.
Dams E, Hendriks L, Van de Peer Y, Neefs J-M, Smits G, Vandenbempt I, de Wachter R. Compilation of small ribosomal subunit RNA sequences. Nucl Acids Res suppl 1988; 16: v87–v173.
Afseth G, Mo Y-Y, Malavia LP. Characterization of the 23S and 5S rRNA genes of Coxiella burnetii and identification of an intervening sequence within the 23S rRNA gene. J Bacteriol 1995; 177: 2946–2949.
Stein A, Saunders NA, Taylor AG, Raoult D. Phylogenic homogeneity of Coxiella burnetii strains as determined by 16S ribosomal RNA sequencing. FEMS Microbiology Letters 1993; 113: 339–344.
Thiele D, Willems H, Krauss H. The 16S/23S ribosomal spacer region of Coxiella burnetti. Eur J Epidemiol 1994; 10: 421–426.
Weisburg WG, Dobson ME, Samuel JE, Dasch GA, Mallavia LP, Baca O, Mandelco L, Sechrest JE, Weiss E, Woese CR. Phylogenetic diversity of the Rickettsiae. J Bacteriol 1989; 171: 4202–4206.
Vene S. Diagnosis of rickettsial infections in Swedish patients: Detection of specific IgM. Eur J Epidemiol 1989; 5: 436–437.
Stein A, Raoult D. Detection of Coxiella burnetii by DNA amplification using polymerase chain reaction. J Clin Microbiol 1992; 30: 2462–2466.
Willems H, Thiele D, Frölich-Ritter R, Krauss H. Detection of Coxiella burnetii in cow's milk using the polymerase chain reaction (PCR). J Vet Med B 1994; 41: 580–587.
Sambrook J, Fritch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989.
Lane D.J. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds), Nucleic Acid Techniques in Bacterial Systematics, 1st edn. New York: John Wiley & Sons. 1991; 115: 175–193.
Boo R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-van Dillen PME, van der Nordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990; 28: 495–503.
Ralph D, McClelland M. Intervening sequence with conserved open reading frame in eubacterial 23S rRNA genes. Proc Natl Acad Sci USA 1993; 90: 6864–6868.
Burgin AB, Parodos K, Lane DJ, Pace NR. The excision of intervening sequences from Salmonella 23S ribosomal RNA. Cell 1990; 4: 405–414.
Skurnik M, Toivanen P. Intervening sequences (IVSs) in the 23S ribosomal RNA genes of pathogenic Yersinia enterocolitica strains: The IVSs in Y. enterocolitica and Salmonella typhimurium have a common origin. Mol Microbiol 1991; 5: 585–593.
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Ibrahim, A., Norlander, L., Macellaro, A. et al. Specific detection of Coxiella burnetii through partial amplification of 23S rDNA. Eur J Epidemiol 13, 329–334 (1997). https://doi.org/10.1023/A:1007385104687
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DOI: https://doi.org/10.1023/A:1007385104687