16S rDNA PCR and denaturing gradient gel electrophoresis; a single generic test for detecting and differentiating species Free

Abstract

Diagnosis of infection is normally based on culture and serological tests, which can be time-consuming and laborious. A number of specific PCRs have been developed but to date there has not been a single generic test capable of detecting and differentiating mycoplasmas to a species level. This report describes the development of a new diagnostic test based on PCR of the 16S rRNA gene with -specific primers and separation of the PCR product according to primary sequence using denaturing gradient gel electrophoresis (DGGE). DGGE enabled the differentiation of 67 species of human and veterinary origin and represents a significant improvement on current tests as diagnosis of infection can be made directly from clinical samples in less than 24 h.

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2005-08-01
2024-03-28
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References

  1. Ashelford K. E., Weightman A. J., Fry J. C. 2002; PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database. Nucleic Acids Res 30:3481–3489 [CrossRef]
    [Google Scholar]
  2. Chalker V. J., Brownlie J. 2004; Taxonomy of the canine Mollicutes by 16S rRNA gene and 16S/23S rRNA intergenic spacer region sequence comparison. Int J Syst Evol Microbiol 54:537–542 [CrossRef]
    [Google Scholar]
  3. Domann E., Hong G., Imirzalioglu C., Turschner S., Kuhle J., Watzel C., Hain T., Hossain H., Chakraborty T. 2003; Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients. J Clin Microbiol 41:5500–5510 [CrossRef]
    [Google Scholar]
  4. Fischer S. G., Lerman L. S. 1983; DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with theory. Proc Natl Acad Sci U S A 80:1579–1583 [CrossRef]
    [Google Scholar]
  5. Heldtander M., Wesonga H., Bolske G., Pettersson B., Johansson K. E. 2001; Genetic diversity and evolution of Mycoplasma capricolum subsp. capripneumoniae strains from eastern Africa assessed by 16S rDNA sequence analysis. Vet Microbiol 78:13–28 [CrossRef]
    [Google Scholar]
  6. Hurtle W., Bode E., Kaplan R. S., Garrison J., Kearney B., Shoemaker D., Henchal E., Norwood D. 2003; Use of denaturing high-performance liquid chromatography to identify Bacillus anthracis by analysis of the 16S-23S rRNA interspacer region and gyrA gene. J Clin Microbiol 41:4758–4766 [CrossRef]
    [Google Scholar]
  7. Jensen J. S., Borre M. B., Dohn B. 2003; Detection of Mycoplasma genitalium by PCR amplification of the 16S rRNA gene. J Clin Microbiol 41:261–266 [CrossRef]
    [Google Scholar]
  8. Lerman L. S., Beldjord C. 1999; Comprehensive mutation detection with denaturing gradient gel electrophoresis. In Mutation Detection pp 35–61 Edited by Cotton R. G. H, Edkins E, Forrest S. New York: Oxford University Press;
    [Google Scholar]
  9. McAuliffe L., Ellis R. J., Ayling R. D., Nicholas R. A. 2003; Differentiation of Mycoplasma species by 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis fingerprinting. J Clin Microbiol 41:4844–4847 [CrossRef]
    [Google Scholar]
  10. Muyzer G. 1999; DGGE/TGGE: a method for identifying genes from natural ecosystems. Curr Opin Microbiol 2:317–322 [CrossRef]
    [Google Scholar]
  11. Muyzer G., de Waal E. C., Uitterlinden A. G. 1993; Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700
    [Google Scholar]
  12. Nicholas R., Baker S. 1998; Recovery of mycoplasmas from animals. Methods Mol Biol 104:37–43
    [Google Scholar]
  13. Ogilvie T. H., Rosendal S., Blackwell T. E., Rostkowski C. M., Julian R. J., Ruhnke L. 1983; Mycoplasma felis as a cause of pleuritis in horses. J Am Vet Med Assoc 182:1374–1376
    [Google Scholar]
  14. Pettersson B., Leitner T., Ronaghi M., Bolske G., Uhlen M., Johansson K. E. 1996; Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons. J Bacteriol 178:4131–4142
    [Google Scholar]
  15. Pettersson B., Tully J. G., Bolske G., Johansson K. E. 2001; Re-evaluation of the classical Mycoplasma lipophilum cluster (Weisburg et al .1989) and description of two new clusters in the hominis group based on 16S rDNA sequences. Int J Syst Evol Microbiol 51:633–643
    [Google Scholar]
  16. Stellrecht K. A., Woron A. M., Mishrik N. G., Venezia R. A. 2004; Comparison of multiplex PCR assay with culture for detection of genital mycoplasmas. J Clin Microbiol 42:1528–1533 [CrossRef]
    [Google Scholar]
  17. Wang H., Kong F., Jelfs P., James G., Gilbert G. L. 2004; Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization. Appl Environ Microbiol 70:1483–1486 [CrossRef]
    [Google Scholar]
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