A PCR-DNA probe assay specific for Bacteroides forsythus.
Bacteroides forsythus is a fastidious anaerobic Gram-negative organism associated with active periodontal disease. The ability of random amplified polymorphic DNA (RAPD) fingerprinting to generate species-specific markers was exploited towards the construction of a polymerase chain reaction (PCR)-DNA probe assay specific for B. forsythus. The strategy included the four following steps: (1) construction of a first generation DNA probe based on a 507-bp RAPD species-specific marker; (2) cloning and sequencing the 507-bp RAPD marker; (3) design of the primer pair Bf 392-1/Bf 392-2 flanking a 392-bp specific internal sequence; and (4) synthesis of quantities of a 392-bp second generation DNA probe by PCR amplification. The PCR-DNA probe assay includes a PCR amplification of a 392-bp specific sequence in the genomic DNA of B. forsythus strains followed by hybridization with the 392-bp digoxigenin-labelled second generation probe. We observed strong, specific hybridization with the amplified DNAs from 11 stains of B. forsythus and no cross-hybridization with the PCR products from 22 foreign species. The PCR-DNA probe assay must be seen as a highly specific and sensitive method for the detection of B. forsythus in mixed infections.
Guillot
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E
Groupe de Recherche en Ecologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec, Canada.
Mouton
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C
D001439:Bacteroides / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D016172:DNA Fingerprinting; D017931:DNA Primers; D015342:DNA Probes / Q000096:biosynthesis / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005819:Genetic Markers; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D019105:Random Amplified Polymorphic DNA Technique; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D005819:Genetic Markers