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 amp lified polymorphic DNA (RAPD) fingerprinting to generate species-speci fic markers was exploited towards the construction of a polymerase cha in reaction (PCR)-DNA probe assay specific for B. forsythus. The strat egy included the four following steps: (1) construction of a first gen eration DNA probe based on a 507-bp RAPD species-specific marker; (2) cloning and sequencing the 507-bp RAPD marker; (3) design of the prime r 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 pro be by PCR amplification. The PCR-DNA probe assay includes a PCR amplif ication of a 392-bp specific sequence in the genomic DNA of B. forsyth us strains followed by hybridization with the 392-bp digoxigenin-label led second generation probe. We observed strong, specific hybridizatio n with the amplified DNAs from 11 stains of B. forsythus and no cross- hybridization with the PCR products from 22 foreign species. The PCR-D NA probe assay must be seen as a highly specific and sensitive method for the detection of B. forsythus in mixed infections. (C) 1996 Academ ic Press Limited