Whole genomic DNA probe for detection of Porphyromonas endodontalis.
The purpose of the present study was to develop a DNA probe for Porphyromonas endodontalis. Pure cultures of P. endodontalis were grown in TYP medium, in an anaerobic chamber. DNA was extracted from the P. endodontalis and labeled using the Genius System by Boehringer Mannheim. The labeled P. endodontalis DNA was used in dot-blot hybridization reactions with homologous (P. endodontalis) and unrelated bacterial samples. To determine specificity, strains of 40 other oral bacterial species (e.g. Porphyromonas gingivalis, Porphyromonas asaccharolytica, and Prevotella intermedia) were spotted and reacted with the P. endodontalis DNA probe. None of the panel of 40 oral bacteria hybridized with the P. endodontalis probe, whereas the blot of the homologous organism showed a strong positive reaction. To determine the sensitivity of the probe, dilutions of a P. endodontalis suspension of known concentration were blotted onto a nylon membrane and reacted with the probe. The results of our investigation indicate that the DNA probe that we have prepared specifically detects only P. endodontalis and can detect at least 3 x 10(4) cells.
Nissan
R
R
Department of Endodontology, Temple University Dental School, 3223 North Broad Street, Philadelphia, PA 19140-5096, USA.
Makkar
S R
SR
Sela
M N
MN
Stevens
R
R
D015169:Colony Count, Microbial; D015342:DNA Probes / Q000138:chemical synthesis; D004269:DNA, Bacterial / Q000235:genetics; D016680:Genome, Bacterial; D006801:Humans; D015151:Immunoblotting; D009693:Nucleic Acid Hybridization; D017861:Porphyromonas / Q000145:classification* / Q000235:genetics; D016966:Porphyromonas gingivalis / Q000235:genetics; D018713:Prevotella intermedia / Q000235:genetics; D013045:Species Specificity
D015342:DNA Probes; D004269:DNA, Bacterial