Development and Laboratory Evaluation of a porA-Targeting TaqMan qPCR Assay for Neisseria gonorrhoeae: Clinical Specimen Validation and Molecular Estimation Using Cultured Reference Strains

Neisseria gonorrhoeae is a major sexually transmitted pathogen and global public health concern. A porA pseudogene-targeting TaqMan quantitative PCR (qPCR) assay was evaluated as a complementary laboratory-based method for the qualitative detection of N. gonorrhoeae and exploratory Z017-calibrated molecular estimation in cultured reference strains. Two candidate probe sets were evaluated, and the probe set generating a 157 bp amplicon was selected for a defined two-step qPCR workflow with a 52 min on-instrument amplification runtime. In silico analysis showed compatible target site arrangements in all 201 N. gonorrhoeae genomes and none in 114 non-target organisms. The assay detected N. gonorrhoeae DNA down to 1 × 10−5 ng/µL, with no amplification of the tested non-target organisms. All 23 N. gonorrhoeae reference strains and 80 N. gonorrhoeae-positive clinical specimens were detected, whereas no amplification was observed in the 20 negative clinical specimens, using DNA extracted from the primary clinical specimens. A standard curve was generated from the titered Z017 reference material (R2 = 0.999; efficiency, 94.6%), which enabled reproducible qPCR-derived CFU/mL-equivalent molecular estimation in cultured World Health Organization reference strains. The qPCR-derived estimates were lower than the paired culture-derived CFU/mL values, and the paired log10-transformed values did not show a statistically significant correlation. These findings provide an assay design and validation-oriented laboratory-based framework combining in silico sequence assessment, the qualitative detection of N. gonorrhoeae in clinical specimens, and exploratory molecular estimation in cultured reference strains, supporting future assay development and bacterial burden research.