PCR-dot blot hybridization based on the neuraminidase-encoding gene is useful for detection of Bacteroides fragilis.
Bacteroides fragilis is a Gram-negative obligate anaerobe frequently isolated from clinical specimens and sometimes causes severe septicemia in compromised hosts. Increasing interest has been shown in the enterotoxigenicity and drug resistance of B. fragilis in the field of medical microbiology. We previously reported rapid detection of this anaerobe by nested PCR targeting a neuraminidase-encoding gene nanH. In the present study, we synthesized a digoxigenin-labeled oligonucleotide probe, NH1, which is specific for nanH of B. fragilis, and we combined the hybridization assay using NH1 with the nanH-PCR to detect this anaerobe in a bacteremia model mice. In the specificity test, the oligonucleotide probe, NH1, hybridized only to amplification products from B. fragilis. PCR-dot blot hybridization based on nanH enabled detection of cells of B. fragilis in blood samples even when the number was as low as 2 x 10(3) colony-forming units/ml. These findings suggest that PCR-dot blot hybridization targeting nanH is a useful procedure for diagnosis of septicemia caused by B. fragilis when viable cells in blood cannot be detected by the traditional culture techniques.
Kuwahara
T
T
Department of Bacteriology, University of Tokushima School of Medicine, Tokushima, Japan.
Nakayama
H
H
Miki
T
T
Kataoka
K
K
Arimochi
H
H
Ohnishi
Y
Y
D000818:Animals; D001442:Bacteroides Infections / Q000382:microbiology*; D001441:Bacteroides fragilis / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D005798:Genes, Bacterial; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000032:analysis / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods
D009439:Neuraminidase