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PCR-DNA probe assay for Bacteroides forsythus

Christian Mouton Christian.Mouton at greb.ulaval.ca
Wed Dec 4 10:24:31 EST 1996


The following paper will be published in the December 1996 issue of
Molecular and Cellular Probes:

A PCR-DNA  probe assay specific for Bacteroides forsythus
Emmanuelle Guillot and Christian Mouton

Abstract
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 strains 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.

Christian Mouton, DCD, DSO
Groupe de Recherche en Ecologie Buccale
Faculte de medecine dentaire, Universite Laval
Quebec (Quebec) G1K 7P4  CANADA
tel. (418)656-5872;  fax. (418)656-2861









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