Evaluation of a fluorescent DNA hybridization assay for the detection ofNeisseria 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.

Multiplex PCR for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Genitourinary specimens

“…VOL. 33, 1995 NOTES 3051 lacked sensitivity (5,6,24). Recent studies of nucleic acid amplification technologies such as PCR and ligase chain reaction have indicated that FVU specimens may be the preferred specimens for diagnosing C. trachomatis infections in both men and women.…”

Development of a Magnetic Microplate Chemifluorimmunoassay for Rapid Detection of Bacteria and Toxin in Blood

“…More importantly it has a large Stoke's shift (130 nm) that minimizes the possible autofluorescence from unwashed sample residues. It has been reported that the AttoPhos has an attomolar detection level for AP molecules and a picogram sensitivity for DNA detection (13,14). Because of the large turnover rate when an excess of AttoPhos as the substrate is used, the dynamic reaction permits the maximum chemifluorescence within 10 min to 1 h according the manufacturer.…”