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Detection of Neisseria gonorrhoeae by commercial and in-house–based assays has been hampered by false-positive and false-negative results. The current study describes a sensitive and specific real-time 5′-nuclease PCR assay targeting a 90-bp region of the multicopy opa gene.To evaluate the sensitivity and specificity of this assay in detection of gonococcus.Sensitivity and specificity were determined by testing a panel of 173 microorganisms. In addition, 135 clinical samples previously evaluated by 4 nucleic acid amplification methods were also tested.A sensitivity of 1 copy per reaction was achieved. Positive results were only obtained for N gonorrhoeae strains including 20 cppB-negative strains. Overall, 134 of 135 clinical sample results agreed with the consensus nucleic amplification methods.This study demonstrates opa-based target can be used as an accurate and rapid PCR assay for the detection of N gonorrhoeae in clinical specimens.