A PCR-DNA probe assay specific forBacteroides forsythus

Bacteroides forsythusis 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 forB. 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 ofB. forsythusstrains followed by hybridization with the 392-bp digoxigenin-labelled second generation probe. We observed strong, specific hybridization with the amplified DNAs from 11 stains ofB. forsythusand 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 ofB. forsythusin mixed infections.