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Gc vaccines have been hindered by Gc\u2019s ability to evade immune surveillance in part by varying its major surface antigens like the type IV pilus. We developed a quick and precise method for measuring pilin antigenic variation (Av) frequency using droplet digital PCR (ddPCR) technology. Two fluorescent probes were designed to detect either the hypervariable tail region of silent pilin locus\n              <jats:italic toggle=\"yes\">pilS<\/jats:italic>\n              3-copy 1 (S3C1) or a sequence conserved in all\n              <jats:italic toggle=\"yes\">pilE<\/jats:italic>\n              variants (CYS2). The appropriate frequency of pilin antigenic variation is measured by the proportion of\n              <jats:italic toggle=\"yes\">pilE<\/jats:italic>\n              amplicons carrying the recombinant S3C1 copy relative to the total pilE amplicons measured by CYS2. The ddPCR assay is specific for RecA-dependent pilin antigenic variation. The reduced frequency of pilin Av in strains lacking RecA-modulating recombination protein RecX and the DNA helicase RecQ confirms the ability of the assay to measure changes in pilin Av frequency. We used the ddPCR assay to determine that pilin Av frequency is altered by the colony densities on a solid medium. The ddPCR assay is an accurate, efficient way to measure Gc pilin Av frequency.\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>IMPORTANCE<\/jats:title>\n            <jats:p>\n              Gonorrhea is a sexually transmitted infectious disease of the human genital and nasopharyngeal mucosa caused by the host-restricted bacterium\n              <jats:italic toggle=\"yes\">Neisseria gonorrhoeae<\/jats:italic>\n              . The rise of antibiotic-resistant gonorrhea is an urgent global threat to public health. Pilus antigenic variation is a gene conversion process that allows\n              <jats:italic toggle=\"yes\">N. gonorrhoeae<\/jats:italic>\n              to evade host immune surveillance, and a mechanistic understanding of this process is crucial to understanding\n              <jats:italic toggle=\"yes\">N. gonorrhoeae<\/jats:italic>\n              pathogenesis. This report shows that we can adopt a digital PCR methodology to quickly and accurately measure pilin antigenic variation.\n            <\/jats:p>\n          <\/jats:sec>","DOI":"10.1128\/msphere.00094-25","type":"journal-article","created":{"date-parts":[[2025,4,23]],"date-time":"2025-04-23T13:00:12Z","timestamp":1745413212000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["A droplet digital PCR assay to measure pilin antigenic variation frequency in\n            <i>Neisseria gonorrhoeae<\/i>"],"prefix":"10.1128","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2164-3198","authenticated-orcid":false,"given":"Sarah J.","family":"Quillin","sequence":"first","affiliation":[{"name":"Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine","place":["Chicago, USA"]},{"name":"Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine","place":["Chicago, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Di","family":"Luo","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine","place":["Chicago, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aoife","family":"Gavagan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine","place":["Chicago, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-2804-3077","authenticated-orcid":true,"given":"Arthur","family":"Prindle","sequence":"additional","affiliation":[{"name":"Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine","place":["Chicago, USA"]},{"name":"Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine","place":["Chicago, USA"]},{"name":"Center for Synthetic Biology, Northwestern University","place":["Evanston, USA"]},{"name":"Department of Chemical and Biological Engineering, Northwestern University","place":["Evanston, USA"]},{"name":"Chan Zuckerberg Biohub Chicago","place":["Chicago, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8651-0375","authenticated-orcid":true,"given":"H Steven","family":"Seifert","sequence":"additional","affiliation":[{"name":"Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine","place":["Chicago, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","volume-title":"World health organization bacterial priority pathogens list, 2024: bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance","author":"WHO","year":"2024","unstructured":"WHO. 2024. 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