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. 1997 Jul;35(7):1876–1882. doi: 10.1128/jcm.35.7.1876-1882.1997

PCR-DNA probe assays for identification and detection of Prevotella intermedia sensu stricto and Prevotella nigrescens.

E Guillot 1, C Mouton 1
PMCID: PMC229862  PMID: 9196214

Abstract

The purpose of this study was to construct PCR-DNA probe assays specific for Prevotella intermedia sensu stricto and Prevotella nigrescens based on the ability of randomly amplified polymorphic DNA (RAPD) fingerprinting to generate species-specific markers. The strategy included four steps: (i) construction of first-generation DNA probes from a 850-bp RAPD marker for P. intermedia sensu stricto and a 1,300-bp RAPD marker for P. nigrescens, (ii) cloning and sequencing of each RAPD marker, (iii) designing of primer pairs flanking specific internal sequences of 754 bp for P. intermedia sensu stricto and of ca. 1,100 bp for P. nigrescens, and (iv) synthesis (by PCR amplification) and digoxigenin labeling of quantities of DNA probes 754 and ca. 1,100 bp in size. The PCR-DNA probe assays combine either PCR amplification of a 754-bp specific sequence in the genomic DNA of strains of P. intermedia sensu stricto and hybridization with the 754-bp digoxigenin-labeled probe or amplification of a ca. 1,100-bp sequence of P. nigrescens and hybridization with the ca. 1,100-bp probe. Specific hybridization was observed with the amplified DNAs from 25 strains of P. intermedia and 24 strains of P. nigrescens, and no reaction was observed with the PCR products from 20 foreign species. The PCR-DNA probe assays described here should allow a highly specific and sensitive detection of P. intermedia sensu stricto and P. nigrescens in mixed infections.

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Selected References

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  1. 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]
  2. Conrads G., Brauner A. Non-radioactively labelled DNA probes for the detection of periodontopathogenic Prevotella and Porphyromonas species. FEMS Immunol Med Microbiol. 1993 Mar;6(2-3):115–120. doi: 10.1111/j.1574-695X.1993.tb00313.x. [DOI] [PubMed] [Google Scholar]
  3. Dahlén G. G., Johnson J. R., Gmür R. Prevotella intermedia and Prevotella nigrescens serotypes, ribotypes and binding characteristics. FEMS Microbiol Lett. 1996 Apr 15;138(1):89–95. doi: 10.1111/j.1574-6968.1996.tb08140.x. [DOI] [PubMed] [Google Scholar]
  4. Dahlén G., Wikström M., Renvert S., Gmür R., Guggenheim B. Biochemical and serological characterization of Bacteroides intermedius strains isolated from the deep periodontal pocket. J Clin Microbiol. 1990 Oct;28(10):2269–2274. doi: 10.1128/jcm.28.10.2269-2274.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Devine D. A., Pearce M. A., Gharbia S. E., Shah H. N., Dixon R. A., Gmür R. Species-specificity of monoclonal antibodies recognising Prevotella intermedia and Prevotella nigrescens. FEMS Microbiol Lett. 1994 Jul 1;120(1-2):99–104. doi: 10.1111/j.1574-6968.1994.tb07014.x. [DOI] [PubMed] [Google Scholar]
  6. Dix K., Watanabe S. M., McArdle S., Lee D. I., Randolph C., Moncla B., Schwartz D. E. Species-specific oligodeoxynucleotide probes for the identification of periodontal bacteria. J Clin Microbiol. 1990 Feb;28(2):319–323. doi: 10.1128/jcm.28.2.319-323.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fani R., Damiani G., Di Serio C., Gallori E., Grifoni A., Bazzicalupo M. Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for microorganisms. Mol Ecol. 1993 Aug;2(4):243–250. doi: 10.1111/j.1365-294x.1993.tb00014.x. [DOI] [PubMed] [Google Scholar]
  8. Frandsen E. V., Poulsen K., Kilian M. Confirmation of the species Prevotella intermedia and Prevotella nigrescens. Int J Syst Bacteriol. 1995 Jul;45(3):429–435. doi: 10.1099/00207713-45-3-429. [DOI] [PubMed] [Google Scholar]
  9. Fukushima H., Moroi H., Inoue J., Onoe T., Ezaki T., Yabuuchi E., Leung K. P., Walker C. B., Clark W. B., Sagawa H. Phenotypic characteristics and DNA relatedness in Prevotella intermedia and similar organisms. Oral Microbiol Immunol. 1992 Feb;7(1):60–64. doi: 10.1111/j.1399-302x.1992.tb00023.x. [DOI] [PubMed] [Google Scholar]
  10. Gharbia S. E., Haapasalo M., Shah H. N., Kotiranta A., Lounatmaa K., Pearce M. A., Devine D. A. Characterization of Prevotella intermedia and Prevotella nigrescens isolates from periodontic and endodontic infections. J Periodontol. 1994 Jan;65(1):56–61. doi: 10.1902/jop.1994.65.1.56. [DOI] [PubMed] [Google Scholar]
  11. Giesendorf B. A., van Belkum A., Koeken A., Stegeman H., Henkens M. H., van der Plas J., Goossens H., Niesters H. G., Quint W. G. Development of species-specific DNA probes for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari by polymerase chain reaction fingerprinting. J Clin Microbiol. 1993 Jun;31(6):1541–1546. doi: 10.1128/jcm.31.6.1541-1546.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gmür R., Guggenheim B. Antigenic heterogeneity of Bacteroides intermedius as recognized by monoclonal antibodies. Infect Immun. 1983 Nov;42(2):459–470. doi: 10.1128/iai.42.2.459-470.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gmür R., Guggenheim B. Interdental supragingival plaque--a natural habitat of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, and Prevotella nigrescens. J Dent Res. 1994 Aug;73(8):1421–1428. doi: 10.1177/00220345940730080501. [DOI] [PubMed] [Google Scholar]
  14. Gmür R. Human serum antibodies against Bacteroides intermedius. Antigenic heterogeneity impairs the interpretation of the host response. J Periodontal Res. 1985 Sep;20(5):492–496. doi: 10.1111/j.1600-0765.1985.tb00832.x. [DOI] [PubMed] [Google Scholar]
  15. Guillot E., Mouton C. A PCR-DNA probe assay specific for Bacteroides forsythus. Mol Cell Probes. 1996 Dec;10(6):413–421. doi: 10.1006/mcpr.1996.0057. [DOI] [PubMed] [Google Scholar]
  16. Haffajee A. D., Socransky S. S. Microbial etiological agents of destructive periodontal diseases. Periodontol 2000. 1994 Jun;5:78–111. doi: 10.1111/j.1600-0757.1994.tb00020.x. [DOI] [PubMed] [Google Scholar]
  17. Könönen E. Pigmented Prevotella species in the periodontally healthy oral cavity. FEMS Immunol Med Microbiol. 1993 Mar;6(2-3):201–205. doi: 10.1111/j.1574-695X.1993.tb00327.x. [DOI] [PubMed] [Google Scholar]
  18. Maiden M. F., Tanner A., Macuch P. J. Rapid characterization of periodontal bacterial isolates by using fluorogenic substrate tests. J Clin Microbiol. 1996 Feb;34(2):376–384. doi: 10.1128/jcm.34.2.376-384.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Martínez-Murcia A. J., Rodríguez-Valera F. The use of arbitrarily primed PCR (AP-PCR) to develop taxa specific DNA probes of known sequence. FEMS Microbiol Lett. 1994 Dec 15;124(3):265–269. doi: 10.1111/j.1574-6968.1994.tb07295.x. [DOI] [PubMed] [Google Scholar]
  20. Moore W. E., Moore L. H., Ranney R. R., Smibert R. M., Burmeister J. A., Schenkein H. A. The microflora of periodontal sites showing active destructive progression. J Clin Periodontol. 1991 Nov;18(10):729–739. doi: 10.1111/j.1600-051x.1991.tb00064.x. [DOI] [PubMed] [Google Scholar]
  21. Moore W. E., Moore L. V. The bacteria of periodontal diseases. Periodontol 2000. 1994 Jun;5:66–77. doi: 10.1111/j.1600-0757.1994.tb00019.x. [DOI] [PubMed] [Google Scholar]
  22. Mättö J., Saarela M., von Troil-Lindén B., Alaluusua S., Jousimies-Somer H., Asikainen S. Similarity of salivary and subgingival Prevotella intermedia and Prevotella nigrescens isolates by arbitrarily primed polymerase chain reaction. Oral Microbiol Immunol. 1996 Dec;11(6):395–401. doi: 10.1111/j.1399-302x.1996.tb00201.x. [DOI] [PubMed] [Google Scholar]
  23. Mättö J., Saarela M., von Troil-Lindén B., Könönen E., Jousimies-Somer H., Torkko H., Alaluusua S., Asikainen S. Distribution and genetic analysis of oral Prevotella intermedia and Prevotella nigrescens. Oral Microbiol Immunol. 1996 Apr;11(2):96–102. doi: 10.1111/j.1399-302x.1996.tb00342.x. [DOI] [PubMed] [Google Scholar]
  24. Ménard C., Gosselin P., Duhaime J. F., Mouton C. Polymerase chain reaction using arbitrary primer for the design and construction of a DNA probe specific for Porphyromonas gingivalis. Res Microbiol. 1994 Oct;145(8):595–602. doi: 10.1016/0923-2508(94)90076-0. [DOI] [PubMed] [Google Scholar]
  25. Pearce M. A., Dixon R. A., Gharbia S. E., Shah H. N., Devine D. A. Characterization of Prevotella intermedia and Prevotella nigrescens by enzyme production, restriction endonuclease and ribosomal RNA gene restriction analyses. Oral Microbiol Immunol. 1996 Jun;11(3):135–141. doi: 10.1111/j.1399-302x.1996.tb00348.x. [DOI] [PubMed] [Google Scholar]
  26. Preus H. R., Russell D. T. Use of a nonradioactive genetic probe identified, synthesized, and labeled in the polymerase chain reaction. Scand J Dent Res. 1994 Jun;102(3):161–167. doi: 10.1111/j.1600-0722.1994.tb01173.x. [DOI] [PubMed] [Google Scholar]
  27. Shah H. N., Collins D. M. Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int J Syst Bacteriol. 1990 Apr;40(2):205–208. doi: 10.1099/00207713-40-2-205. [DOI] [PubMed] [Google Scholar]
  28. Shah H. N., Gharbia S. E. Biochemical and chemical studies on strains designated Prevotella intermedia and proposal of a new pigmented species, Prevotella nigrescens sp. nov. Int J Syst Bacteriol. 1992 Oct;42(4):542–546. doi: 10.1099/00207713-42-4-542. [DOI] [PubMed] [Google Scholar]
  29. Shah H. N., Gharbia S. E., Scully C., Finegold S. M. Oligonucleotide probes to the 16S ribosomal RNA: implications of sequence homology and secondary structure with particular reference to the oral species Prevotella intermedia and Prevotella nigrescens. Oral Dis. 1995 Mar;1(1):32–36. doi: 10.1111/j.1601-0825.1995.tb00154.x. [DOI] [PubMed] [Google Scholar]
  30. Slots J., Listgarten M. A. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases. J Clin Periodontol. 1988 Feb;15(2):85–93. doi: 10.1111/j.1600-051x.1988.tb00999.x. [DOI] [PubMed] [Google Scholar]
  31. Welsh J., McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990 Dec 25;18(24):7213–7218. doi: 10.1093/nar/18.24.7213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Williams J. G., Kubelik A. R., Livak K. J., Rafalski J. A., Tingey S. V. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990 Nov 25;18(22):6531–6535. doi: 10.1093/nar/18.22.6531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. van Steenbergen T. J., Timmerman M. F., Mikx F. H., de Quincey G., van der Weijden G. A., van der Velden U., de Graaff J. Discrepancy between culture and DNA probe analysis for the detection of periodontal bacteria. J Clin Periodontol. 1996 Oct;23(10):955–959. doi: 10.1111/j.1600-051x.1996.tb00518.x. [DOI] [PubMed] [Google Scholar]

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