title: Evaluation of a fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae.
abstract: This study evaluates a four-hour fluorescent DNA hybridization assay using both known bacterial isolates and clinical specimens. A biotinylated oligonucleotide probe from a sequence of the plasmid-encoded gene cppB was used. Hybrids were detected by addition of a streptavidin-alkaline phosphatase conjugate, followed by incubation for 30 min in a fluorescent substrate for alkaline phosphatase. The level of detection of the fluorescent assay was 0.1 pg of cryptic plasmid DNA or 200 cfu of the plasmid-containing strain NG 34/85 of Neisseria gonorrhoeae. A total of 119 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All Neisseria gonorrhoeae strains, including eight plasmid-free strains, hybridized with the probe. Fluorescence ratios were 2.67 for plasmid-free strains and 3.85 for plasmid-containing strains. Of the heterologous microorganisms tested, only one of six strains of Neisseria cinerea gave a fluorescence ratio above the 2.0 cut-off value for positivity with the probe at a cell density of 1 x 10(4) cfu. The probe was also evaluated using clinical specimens from 100 patients attending a clinic for sexually transmitted diseases. The sensitivity of the assay was 100% while the specificity was 97.5%. Positive and negative predictive values were 91.2% and 100%, respectively. The fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae described here thus appears to be a highly specific and sensitive assay.
authors.affiliation: Biological Sciences Department, California Polytechnic State University, San Luis Obispo 93407.
authors.forename: R J
authors.initials: RJ
authors.lastname: Cano
authors.forename: J C
authors.initials: JC
authors.lastname: Palomares
authors.forename: M J
authors.initials: MJ
authors.lastname: Torres
authors.forename: R E
authors.initials: RE
authors.lastname: Klem
mesh_terms: D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D005453:Fluorescence; D006801:Humans; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D010957:Plasmids
chemical_list: D004269:DNA, Bacterial