Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1997 Nov;35(11):2854–2858. doi: 10.1128/jcm.35.11.2854-2858.1997

Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions.

P H Hendolin 1, A Markkanen 1, J Ylikoski 1, J J Wahlfors 1
PMCID: PMC230074  PMID: 9350746

Abstract

A multiplex PCR procedure was developed for the simultaneous detection of Alloiococcus otitidis, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in middle ear effusions (MEEs) from patients with chronic otitis media with effusion. The bacterial 16S rRNA gene was chosen as the target, and the procedure used one common lower primer and four species-specific upper primers. The reaction was optimized by changing the primer concentrations to yield equal amounts of amplification products. The specificity of the reaction was verified with various bacterial species found in the nasopharynx. The performance of the procedure was examined with 25 MEE specimens, and the results were compared to those obtained by conventional culture methods. A detection level of 10 bacterial cells/reaction for each of the study organisms was achieved. By conventional culture methods, 8 (32%) of the specimens showed growth of one of the study organisms. In contrast, 21 (84%) of the specimens tested positive by the multiplex PCR. None of the culture-positive specimens were PCR negative, whereas three (12%) of the PCR-positive specimens tested positive for two of the four study organisms. Thus, the multiplex PCR method improves the detection rate significantly compared to that of the conventional culture method.

Full Text

The Full Text of this article is available as a PDF (1,006.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aguirre M., Collins M. D. Development of a polymerase chain reaction-probe test for identification of Alloiococcus otitis. J Clin Microbiol. 1992 Aug;30(8):2177–2180. doi: 10.1128/jcm.30.8.2177-2180.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Bluestone C. D. Management of otitis media in infants and children: current role of old and new antimicrobial agents. Pediatr Infect Dis J. 1988 Nov;7(11 Suppl):S129–S136. doi: 10.1097/00006454-198811001-00002. [DOI] [PubMed] [Google Scholar]
  4. Bosley G. S., Whitney A. M., Pruckler J. M., Moss C. W., Daneshvar M., Sih T., Talkington D. F. Characterization of ear fluid isolates of Alloiococcus otitidis from patients with recurrent otitis media. J Clin Microbiol. 1995 Nov;33(11):2876–2880. doi: 10.1128/jcm.33.11.2876-2880.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Edwards M. C., Gibbs R. A. Multiplex PCR: advantages, development, and applications. PCR Methods Appl. 1994 Feb;3(4):S65–S75. doi: 10.1101/gr.3.4.s65. [DOI] [PubMed] [Google Scholar]
  6. Faden H., Dryja D. Recovery of a unique bacterial organism in human middle ear fluid and its possible role in chronic otitis media. J Clin Microbiol. 1989 Nov;27(11):2488–2491. doi: 10.1128/jcm.27.11.2488-2491.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Giebink G. S. The microbiology of otitis media. Pediatr Infect Dis J. 1989 Jan;8(1 Suppl):S18–S20. [PubMed] [Google Scholar]
  8. Greisen K., Loeffelholz M., Purohit A., Leong D. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol. 1994 Feb;32(2):335–351. doi: 10.1128/jcm.32.2.335-351.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hotomi M., Tabata T., Kakiuchi H., Kunimoto M. Detection of Haemophilus influenzae in middle ear of otitis media with effusion by polymerase chain reaction. Int J Pediatr Otorhinolaryngol. 1993 Aug;27(2):119–126. doi: 10.1016/0165-5876(93)90127-o. [DOI] [PubMed] [Google Scholar]
  10. Meyerhoff W. L., Giebink G. S. Panel discussion: pathogenesis of otitis media. Pathology and microbiology of otitis media. Laryngoscope. 1982 Mar;92(3):273–277. doi: 10.1288/00005537-198203000-00010. [DOI] [PubMed] [Google Scholar]
  11. Miller P. H., Facklam R. R., Miller J. M. Atmospheric growth requirements for Alloiococcus species and related gram-positive cocci. J Clin Microbiol. 1996 Apr;34(4):1027–1028. doi: 10.1128/jcm.34.4.1027-1028.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Palva T., Häyry P., Raunio V., Ylikoski J. Immunologic aspects of otitis media. Acta Otolaryngol. 1980 Mar-Apr;89(3-4):177–181. doi: 10.3109/00016488009127125. [DOI] [PubMed] [Google Scholar]
  13. Post J. C., Preston R. A., Aul J. J., Larkins-Pettigrew M., Rydquist-White J., Anderson K. W., Wadowsky R. M., Reagan D. R., Walker E. S., Kingsley L. A. Molecular analysis of bacterial pathogens in otitis media with effusion. JAMA. 1995 May 24;273(20):1598–1604. [PubMed] [Google Scholar]
  14. Ueyama T., Kurono Y., Shirabe K., Takeshita M., Mogi G. High incidence of Haemophilus influenzae in nasopharyngeal secretions and middle ear effusions as detected by PCR. J Clin Microbiol. 1995 Jul;33(7):1835–1838. doi: 10.1128/jcm.33.7.1835-1838.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Virolainen A., Salo P., Jero J., Karma P., Eskola J., Leinonen M. Comparison of PCR assay with bacterial culture for detecting Streptococcus pneumoniae in middle ear fluid of children with acute otitis media. J Clin Microbiol. 1994 Nov;32(11):2667–2670. doi: 10.1128/jcm.32.11.2667-2670.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wahlfors J., Meurman J. H., Väisänen P., Alakuijala P., Korhonen A., Torkko H., Jänne J. Simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by a rapid PCR method. J Dent Res. 1995 Nov;74(11):1796–1801. doi: 10.1177/00220345950740111301. [DOI] [PubMed] [Google Scholar]
  17. Wang R. F., Cao W. W., Wang H., Johnson M. G. A 16S rRNA-based DNA probe and PCR method specific for Listeria ivanovii. FEMS Microbiol Lett. 1993 Jan 1;106(1):85–92. doi: 10.1111/j.1574-6968.1993.tb05939.x. [DOI] [PubMed] [Google Scholar]
  18. Wilson K. H., Blitchington R. B., Greene R. C. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J Clin Microbiol. 1990 Sep;28(9):1942–1946. doi: 10.1128/jcm.28.9.1942-1946.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES