Abstract
A PCR based, reverse capture checkerboard hybridization methodology was designed to rapidly detect and enumerate oral species of Streptococcus and other genera in plaque samples to assess the bacterial etiology of root surface caries or other oral diseases. The procedure circumvents the need for in vitro bacterial cultivation. Up to 30 reverse capture probes that target regions of 16S rRNA genes are deposited on a nylon membrane in separate horizontal lanes using a MiniSlot apparatus. 16S rRNA genes of DNA from plaque or pure cultures are PCR amplified using a digoxigenin-labeled primer. Hybridizations are performed in vertical channels in a Miniblotter apparatus with digoxigenin-labeled amplicons from up to 45 samples. Consequently, 1350 hybridizations are performed simultaneously using a single membrane. Hybridiza-tion signals are detected using chemifluorescence procedures. Bacterial numbers are determined by analyzing hybridization signals using a Storm system. A genus-specific probe is used to assess the total number of streptococci and universal probes are used to assess total bacterial counts. Use of this procedure enables laboratories to analyze thousands of clinical or environmental samples with many probes in a relatively short period. This methodology has broad range applications in microbiology to monitor population distributions in a given environment.
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References
Bentley RW, Leigh JA (1995). Development of PCRBased hybridization protocol for identification of streptococcal species. J Clin Microbiol 33: 1296–1301.
Bentley RW, Leigh JA, Collins MD (1991). Intrageneric structure of Streptococcus based on comparative analysis of small-subunit rRNA sequences. Intern J System Bacteriol 41: 487–494.
Bentley RW, Leigh JA, Collins MD (1993). Development and use of species-specific oligonucleotide probes for differentiation of Streptococcus uberis and Streptococcus parauberis. J Clin Microbiol 31: 57–60.
Cangelosi GA, Iversen JM, Zuo Y, Oswald TK, Lamont RJ (1994). Oligonucleotide probes for mutans streptococci. Mol Cell Probes 8: 73–80.
Choi BK, Paster BJ, Dewhirst FE, Göbel UB (1994). Diversity of cultivable and uncultivable oral spirochetes from a patient with severe destructive periodontitis. Infect Immun 62: 1889–1895.
Dewhirst FE, Paster BJ (1991). DNA probe analyses for the detection of periodontopathic bacteria in clinical samples. In: Hamada S, Holt SC, McGhee JR (eds), Periodontal disease: Pathogens and Host Immune Responses (pp 367–377). London: Quintessence Publishing Co., Ltd.
Giovannoni S (1991). The polymerase chain reaction, p. In: Stackebrandt E, Goodfellow M (eds), Nucleic Acid Techniques in Bacterial Systematics (pp 177–203). John Wiley & Sons, New York.
Jacobs JA, Schot CS, Bunschoten AE, Schouls LM (1996). Rapid species identification of ‘Streptococcus milleri’ strains by line blot hybridization: Identification of a distinct 16S rRNA population closely related to Streptococcus constellatus. J Clin Microbiol 34: 1717–1721.
Jukes TH, Cantor CR (1969). Evolution of protein molecules. In: Munro HN (ed), Mammalian Protein Metabolism, vol. 3 (pp 21–132).Academic Press, Inc., New York.
Kawamura Y, Hou XG, Sultana F, Miura H, Ezaki T (1995). Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Intern J System Bacteriol 45: 406–408.
Liesack W, Stackebrandt E (1992). Unculturable microbes detected by molecular sequences and probes. Biodiversity Conserv 1: 250–262.
Morrissey DV, Collins ML (1989). Nucleic acid hybridization assays employing dA-tailed capture probes. Single capture methods. Molec Cell Probes 3: 189–207.
Nelms LF, Odelson DA, Whitehead TR, Hespell RB (1995). Differentiation of ruminal and human Streptococcus bovis strains by DNA homology and 16S rRNA probes. Current Microbiol 31: 294–300.
Pace NR, Stahl DA, Lane DJ, Olsen GJ (1986). The analysis of natural microbial populations by ribosomal RNA sequences. Adv Microb Ecol 9: 1–55.
Paster BJ, Dewhirst FE (1988). Phylogeny of campylobacters, wolinellas, Bacteroides gracilis, and Bacteroides ureolyticus by 16S ribosomal ribonucleic acid sequencing. Inter J System Bacteriol 38: 56–62.
Paster BJ, Dewhirst FE, Cooke SM, Fussing V, Poulsen LK, Breznak JA (1996). Phylogeny of notyet-cultured spirochetes from termite guts. Appl Environ Microbiol 62: 347–352.
Saiki RK, Walsh PS, Levenson CH, Erlich HA (1989). Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci 86: 6230–6234.
Saitou N, Nei M (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425.
Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE, Levin AE (1994). ‘Checkerboard’ DNA-DNA hybridization. Biotechniques 17: 788–792.
Stackebrandt E, Liesack W, Goebel BM (1993). Bacterial diversity in a soil sample from a subtropical Australian environment as determined by 16S rDNA analysis. FASEB J 7: 232–236.
van Houte J, Lopman J, Kent R (1994). The predominant cultivable flora of sound and carious human root surfaces. J Dent Res 73: 1727–1734.
van Houte J, Lopman J, Kent R (1996). The final pH of bacteria comprising the predominant flora on sound and carious human root and enamel surfaces. J Dent Res 75: 1008–1014.
Ward DM, Weller R, Bateson MM (1990). 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345: 63–65.
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Paster, B.J., Bartoszyk, I.M. & Dewhirst, F.E. Identification of oral streptococci using PCR-based, reverse-capture, checkerboard hybridization. Methods Cell Sci 20, 223–231 (1998). https://doi.org/10.1023/A:1009715710555
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DOI: https://doi.org/10.1023/A:1009715710555