{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T12:29:00Z","timestamp":1786969740974,"version":"build-2736575974"},"reference-count":42,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T00:00:00Z","timestamp":1786924800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>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 \u00d7 10\u22125 ng\/\u00b5L, 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.<\/jats:p>","DOI":"10.3390\/ijms27167335","type":"journal-article","created":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T12:01:26Z","timestamp":1786968086000},"page":"7335","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Laboratory Evaluation of a porA-Targeting TaqMan qPCR Assay for Neisseria gonorrhoeae: Clinical Specimen Validation and Molecular Estimation Using Cultured Reference Strains"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-2763-3162","authenticated-orcid":false,"given":"Changmi","family":"Oh","sequence":"first","affiliation":[{"name":"Department of Medical Laser, College of Medicine, Dankook University, Cheonan 31116, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin-Ho","family":"Kim","sequence":"additional","affiliation":[{"name":"Biomedical Research Institute, Dankook University Hospital, Cheonan 31116, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,17]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2021). Sexually Transmitted Infections (STIs): Fact Sheet, World Health Organization."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e235","DOI":"10.1016\/S1473-3099(17)30310-9","article-title":"Sexually transmitted infections: Challenges ahead","volume":"17","author":"Unemo","year":"2017","journal-title":"Lancet Infect. Dis."},{"key":"ref_3","unstructured":"World Health Organization (2021). Global Health Sector Strategy on Sexually Transmitted Infections 2016\u20132021: Monitoring and Evaluation Report, World Health Organization."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1","DOI":"10.15585\/mmwr.rr7004a1","article-title":"Sexually transmitted infections treatment guidelines, 2021","volume":"70","author":"Workowski","year":"2021","journal-title":"MMWR Recomm. Rep."},{"key":"ref_5","unstructured":"World Health Organization (2021). Guidelines for the Management of Symptomatic Sexually Transmitted Infections, World Health Organization."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"E19","DOI":"10.1111\/j.1749-6632.2011.06215.x","article-title":"Antibiotic resistance in Neisseria gonorrhoeae: Origin, evolution, and lessons learned for the future","volume":"1230","author":"Unemo","year":"2011","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_7","unstructured":"World Health Organization (2023). Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP): Surveillance Report 2023, World Health Organization."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1128\/CMR.19.1.165-256.2006","article-title":"Real-time PCR in clinical microbiology: Applications for routine laboratory testing","volume":"19","author":"Espy","year":"2006","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_9","first-page":"1","article-title":"Recommendations for the laboratory-based detection of Chlamydia trachomatis and Neisseria gonorrhoeae\u20142014","volume":"63","author":"Papp","year":"2014","journal-title":"MMWR Recomm. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5653","DOI":"10.1128\/JCM.43.11.5653-5659.2005","article-title":"Specific and sensitive detection of Neisseria gonorrhoeae in clinical specimens by real-time PCR","volume":"43","author":"Brouwers","year":"2005","journal-title":"J. Clin. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3","DOI":"10.2353\/jmoldx.2006.050045","article-title":"Nucleic acid amplification testing for Neisseria gonorrhoeae","volume":"8","author":"Whiley","year":"2006","journal-title":"J. Mol. Diagn."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1111\/j.1198-743X.2004.00722.x","article-title":"Real-time PCR in the microbiology laboratory","volume":"10","author":"Mackay","year":"2004","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"20862","DOI":"10.2807\/1560-7917.ES2014.19.30.20862","article-title":"Four treatment failures of pharyngeal gonorrhoea with ceftriaxone (500 mg) or cefotaxime (500 mg), Sweden, 2013 and 2014","volume":"19","author":"Golparian","year":"2014","journal-title":"Euro Surveill."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1586\/14787210.2014.880651","article-title":"Point of care diagnostics for sexually transmitted infections: Perspectives and advances","volume":"12","author":"Gaydos","year":"2014","journal-title":"Expert Rev. Anti Infect. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"107197","DOI":"10.1016\/j.mimet.2025.107197","article-title":"Advances in molecular diagnostics of Neisseria gonorrhoeae","volume":"236","author":"Yu","year":"2025","journal-title":"J. Microbiol. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"548","DOI":"10.2471\/BLT.18.228486","article-title":"Chlamydia, gonorrhoea, trichomoniasis and syphilis: Global prevalence and incidence estimates, 2016","volume":"97","author":"Rowley","year":"2019","journal-title":"Bull. World Health Organ."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1128\/CMR.00010-14","article-title":"Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: Past, evolution, and future","volume":"27","author":"Unemo","year":"2014","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"19874","DOI":"10.2807\/ese.16.21.19874-en","article-title":"False-negative results using Neisseria gonorrhoeae porA pseudogene PCR\u2014A clinical gonococcal isolate with an N. meningitidis porA sequence, Australia, March 2011","volume":"16","author":"Whiley","year":"2011","journal-title":"Euro Surveill."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1111\/j.1600-0463.2005.apm_206.x","article-title":"The porA pseudogene of Neisseria gonorrhoeae--low level of genetic polymorphism and a few, mainly identical, inactivating mutations","volume":"113","author":"Unemo","year":"2005","journal-title":"APMIS"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1046\/j.1365-2958.1998.01101.x","article-title":"A gonococcal porA pseudogene: Implications for understanding the evolution and pathogenicity of Neisseria gonorrhoeae","volume":"30","author":"Feavers","year":"1998","journal-title":"Mol. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"574","DOI":"10.2353\/jmoldx.2006.060024","article-title":"A fast real-time polymerase chain reaction method for sensitive and specific detection of the Neisseria gonorrhoeae porA pseudogene","volume":"8","author":"Hjelmevoll","year":"2006","journal-title":"J. Mol. Diagn."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.diagmicrobio.2004.12.011","article-title":"A real-time PCR assay for the detection of Neisseria gonorrhoeae in genital and extragenital specimens","volume":"52","author":"Whiley","year":"2005","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1097\/OLQ.0b013e3181644bc9","article-title":"Clinical validation of a real-time polymerase chain reaction detection of Neisseria gonorrheae porA pseudogene versus culture techniques","volume":"35","author":"Hjelmevoll","year":"2008","journal-title":"Sex. Transm. Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"986","DOI":"10.1101\/gr.6.10.986","article-title":"Real time quantitative PCR","volume":"6","author":"Heid","year":"1996","journal-title":"Genome Res."},{"key":"ref_25","first-page":"1","article-title":"Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field","volume":"116","author":"Aznar","year":"2014","journal-title":"J. Appl. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1128\/jb.1.3.321-331.1916","article-title":"The number of colonies allowable on satisfactory agar plates","volume":"1","author":"Breed","year":"1916","journal-title":"J. Bacteriol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"848","DOI":"10.1016\/j.fm.2011.02.008","article-title":"Recent advances in quantitative PCR (qPCR) applications in food microbiology","volume":"28","author":"Postollec","year":"2011","journal-title":"Food Microbiol."},{"key":"ref_28","first-page":"352","article-title":"Promoting molecular diagnostic equity: Assessing in-house real-time PCR for Neisseria gonorrhoeae in anal samples from MSM recruited in an outpatient setting in Morocco","volume":"32","author":"Qasmaoui","year":"2024","journal-title":"Infez. Med."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e70214","DOI":"10.1111\/1751-7915.70214","article-title":"Clinical validation of loop-mediated isothermal amplification assays for the rapid detection of Neisseria gonorrhoeae and Chlamydia trachomatis","volume":"18","author":"Ahmadi","year":"2025","journal-title":"Microb. Biotechnol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1248\/bpb.b25-00734","article-title":"A pilot study to develop an ultrasensitive Thio-NAD cycling ELISA for Neisseria gonorrhoeae detection","volume":"49","author":"Chen","year":"2026","journal-title":"Biol. Pharm. Bull."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1093\/jac\/dkae446","article-title":"Novel approach using automated target enrichment enables culture-independent accurate whole-genome sequencing of Neisseria gonorrhoeae directly from clinical urogenital and extragenital specimens","volume":"80","author":"Golparian","year":"2025","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Bennett, J.S., Jolley, K.A., Sparling, P.F., Saunders, N.J., Hart, C.A., Feavers, I.M., and Maiden, M.C. (2007). Species status of Neisseria gonorrhoeae: Evolutionary and epidemiological inferences from multilocus sequence typing. BMC Biol., 5.","DOI":"10.1186\/1741-7007-5-35"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1377","DOI":"10.1021\/acsinfecdis.8b00104","article-title":"Direct-qPCR assay for coupled identification and antimicrobial susceptibility testing of Neisseria gonorrhoeae","volume":"4","author":"Chen","year":"2018","journal-title":"ACS Infect. Dis."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Meyer, T., and Buder, S. (2020). The laboratory diagnosis of Neisseria gonorrhoeae: Current testing and future demands. Pathogens, 9.","DOI":"10.3390\/pathogens9020091"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1038\/s41572-019-0128-6","article-title":"Gonorrhoea","volume":"5","author":"Unemo","year":"2019","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1016\/j.mimet.2006.04.015","article-title":"Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells","volume":"67","author":"Nocker","year":"2006","journal-title":"J. Microbiol. Methods"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"804","DOI":"10.2144\/03344rr02","article-title":"Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5\u2032-nuclease PCR","volume":"34","author":"Nogva","year":"2003","journal-title":"BioTechniques"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1997","DOI":"10.1128\/AEM.72.3.1997-2004.2006","article-title":"Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide","volume":"72","author":"Nocker","year":"2006","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_39","unstructured":"World Health Organization (2017). Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP): General Protocol, World Health Organization."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Peris, M.P., Alonso, H., Escolar, C., Tristancho-Bar\u00f3, A., Abad, M.P., Rezusta, A., and Milagro, A. (2024). Detection of Chlamydia trachomatis and Neisseria gonorrhoeae (and Its Resistance to Ciprofloxacin): Validation of a Molecular Biology Tool for Rapid Diagnosis and Treatment. Antibiotics, 13.","DOI":"10.3390\/antibiotics13111011"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1136\/sextrans-2024-056132","article-title":"Molecular Detection of Ceftriaxone Resistance in Neisseria gonorrhoeae Clinical Specimens: A Tool for Public Health Control","volume":"100","author":"Day","year":"2024","journal-title":"Sex. Transm. Infect."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Vasquez, F., Eguiluz, M., Vargas, S.K., Qquellon, J., Caceres, C.F., Klausner, J.D., and Konda, K.A. (2025). Molecular Detection of Mutations in the penA and 23S rRNA Genes of Neisseria gonorrhoeae Related to Decreased Cephalosporin and Azithromycin Susceptibility in Rectal Specimens from Men Who Have Sex with Men (MSM) in Lima, Peru. Trop. Med. Infect. Dis., 10.","DOI":"10.3390\/tropicalmed10080211"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/16\/7335\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T12:12:03Z","timestamp":1786968723000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/16\/7335"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,17]]},"references-count":42,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["ijms27167335"],"URL":"https:\/\/doi.org\/10.3390\/ijms27167335","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,17]]}}}