A new method for rapid screening of bacterial species- or subspecies-specific DNA probes

Abstract

A simple assay for the rapid screening of bacterial species-or subspecies-specific DNA probes for the random cloning method is presented, involving the use of genomic DNAs as probes and recombinant plasmid DNAs containing genomic DNA digested with HindIII as targets. The optimal amount of target DNAs and the concentration of digoxigenin-labeled genomic DNA probes were 20 ng and 100 ng ml 31 (or 10 ng and 200 ng ml 31 ), respectively. The method was applied to the development of Fusobacterium nucleatum subspecies-specific probes. Our results showed that four out of 96 probes were F. nucleatum subspecies-specific, which was confirmed by Southern blot analysis. Our results indicate that the new method can be used for the rapid screening of species-or subspecies-specific probes. ß

Application of rpoB and Zinc Protease Gene for Use in Molecular Discrimination of Fusobacterium nucleatum Subspecies

“…nucleatum ATCC 25586 T (25); the genomic DNA nucleotide sequence of this strain shows that it can produce only three amino acids (glutamate, aspartate, and asparagine) and several amino acids or peptide transporter proteins (18). Considering this, it appears that the zinc protease of F. nucleatum plays a role in dissociating proteins or peptides within the dental plaque matrix, making them available in their cytoplasm.…”

“…nucleatum-specific DNA probe, Fu4, recently was cloned (25). The probe Fu4 (1,268 bp) is composed of the 5Ј end of the partial deoxyuridine 5Ј-triphosphate nucleotidohydrolase (5ЈdUTPase) gene (273 bp out of 441) and the 3Ј end of the partial zinc protease gene (878 bp out of 1,227 bp).…”

Pi30 DNA Probe May Be Useful for the Identification of Prevotella intermedia at the Species or Strain Level

“…2 T by inverted dot blot hybridization (12). The reason for this discrepancy is not immediately clear, but it may be due to the difference in the sensitivity between the two methods.…”

“…Recently, we introduced a new method for rapid screening of bacterial species-or subspecies-specific DNA probes, named the "inverted dot blot hybridization screening method" (12). In our previous study, the inverted dot blot hybridization data showed that a probe, Pi30, was specific for P. intermedia (11).…”

“…Whole genomic DNA was isolated using the method of Lippke et al (14) with a modification (12); bacterial genomic DNAs were purified by the extraction method of phenol/chloroform instead of cesium chloride.…”