{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T06:14:53Z","timestamp":1788243293950,"version":"build-2803163510"},"reference-count":49,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      <jats:italic>Toxoplasma gondii<\/jats:italic>\n                      (\n                      <jats:italic>T. gondii<\/jats:italic>\n                      ) exhibits substantial genetic diversity, and different parasite lineages are associated with distinct epidemiological distributions and biological characteristics. Accurate molecular characterization of\n                      <jats:italic>T. gondii<\/jats:italic>\n                      strains is important for understanding parasite population structure and transmission patterns. However, existing genotyping approaches often require multiple loci, extensive experimental procedures, or complex data analysis. Therefore, simplified and reliable molecular markers for rapid lineage differentiation are still needed.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      In this study, comparative genomic analysis was performed using representative\n                      <jats:italic>T. gondii<\/jats:italic>\n                      strains with well-defined genetic backgrounds and virulence phenotypes. The ROP18 genomic region, including partial genomic sequences, 5\u2032 flanking regions, coding sequence (CDS), and 3\u2032 flanking regions, was analyzed to identify informative polymorphic signatures. A short conserved sequence region containing lineage-associated polymorphic sites was identified within the ROP18 3\u2032 flanking region. Based on these sequence signatures, HRM-PCR and TaqMan MGB probe-based real-time PCR assays were developed and evaluated using plasmid standards and representative\n                      <jats:italic>T. gondii<\/jats:italic>\n                      genomic DNA samples.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Phylogenetic analyses based on different ROP18 genomic regions demonstrated distinct clustering patterns among analyzed strains. Although the ROP18 3\u2032 flanking region was highly conserved, a short conserved sequence region containing informative polymorphic sites was identified, and the combination of these sites generated three distinct lineage-associated ROP18 patterns. Analysis of publicly available genomic datasets further demonstrated that individual strains contained one of these defined patterns rather than multiple patterns simultaneously. The developed HRM-PCR assay successfully discriminated the three ROP18-associated patterns based on distinct melting profiles with good reproducibility. Furthermore, the TaqMan MGB probe-based assay enabled specific identification of different ROP18-associated patterns through defined probe-recognition combinations and showed good analytical performance.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      This study identifies novel lineage-associated molecular signatures within the ROP18 3\u2032 flanking region and establishes complementary HRM-PCR and TaqMan MGB probe-based approaches for rapid molecular differentiation of\n                      <jats:italic>T. gondii<\/jats:italic>\n                      strains. These findings highlight the potential of conserved non-coding regions adjacent to functionally important genes as informative targets for parasite genotyping and provide a practical complementary tool for epidemiological surveillance and strain characterization.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fmicb.2026.1868079","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T05:34:00Z","timestamp":1788240840000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Identification of lineage-associated polymorphisms in the ROP18 3\u2032 flanking region and development of molecular assays for differentiation of Toxoplasma gondii lineages"],"prefix":"10.3389","volume":"17","author":[{"given":"Bao-Yi","family":"Liang","sequence":"first","affiliation":[{"name":"The First Affiliated Hospital\/The First Clinical Medicine School of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia-Li","family":"Liu","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital\/The First Clinical Medicine School of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiu-Mei","family":"Lin","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital\/The First Clinical Medicine School of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi-Hao","family":"Zhang","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital\/The First Clinical Medicine School of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui-Ying","family":"Li","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital\/The First Clinical Medicine School of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing-Hui","family":"Lu","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital\/The First Clinical Medicine School of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei-Jie","family":"Chen","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital\/The First Clinical Medicine School of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zheng","family":"Jian","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital\/The First Clinical Medicine School of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fu-Shi","family":"Quan","sequence":"additional","affiliation":[{"name":"Medical Research Center for Bioreaction to Reactive Oxygen Species and Biomedical Science Institute, Core Research Institute (CRI), Kyung Hee University","place":["Seoul, Republic of Korea"]},{"name":"Department of Medical Zoology, School of Medicine, Kyung Hee University","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi-Ting","family":"Feng","sequence":"additional","affiliation":[{"name":"Department of Pathology, the First Affiliated Hospital of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhou-Rui","family":"Yin","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital\/The First Clinical Medicine School of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhao-Shou","family":"Yang","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital\/The First Clinical Medicine School of Guangdong Pharmaceutical University","place":["Guangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,1]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"4641","DOI":"10.1128\/JCM.01152-10","article-title":"Genotyping of toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay","volume":"48","author":"Ajzenberg","year":"2010","journal-title":"J. Clin. Microbiol."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"588","DOI":"10.1186\/s13071-020-04445-z","article-title":"The life-cycle of toxoplasma gondii reviewed using animations","volume":"13","author":"Attias","year":"2020","journal-title":"Parasit. Vectors"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"106188","DOI":"10.1016\/j.mimet.2021.106188","article-title":"Designing and developing of high-resolution melting technique for separating different types of toxoplasma gondii by analysis of B1 and ROP8 gene regions","volume":"184","author":"Azimpour-Ardakan","year":"2021","journal-title":"J. Microbiol. Methods"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"302","DOI":"10.1016\/j.meegid.2012.12.012","article-title":"Intraspecific comparative genomics of Candida albicans mitochondria reveals non-coding regions under neutral evolution","volume":"14","author":"Bartelli","year":"2013","journal-title":"Infect. Genet. Evol."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"461","DOI":"10.1186\/s13071-023-06080-w","article-title":"Genetic diversity of toxoplasma gondii in South America: occurrence, immunity, and fate of infection","volume":"16","author":"Brito","year":"2023","journal-title":"Parasit. Vectors"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"634","DOI":"10.1093\/clinchem\/hvaf043","article-title":"MIQE 2.0: revision of the minimum information for publication of quantitative real-time PCR experiments guidelines","volume":"71","author":"Bustin","year":"2025","journal-title":"Clin. Chem."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"217","DOI":"10.1186\/s13059-023-03057-x","article-title":"GenArk: towards a million UCSC genome browsers","volume":"24","author":"Clawson","year":"2023","journal-title":"Genome Biol."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"301","DOI":"10.3390\/microorganisms13020301","article-title":"Investigation of virulence-related markers in atypical strains of toxoplasma gondii from Brazil","volume":"13","author":"Costa","year":"2025","journal-title":"Microorganisms"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"250","DOI":"10.1016\/j.ymeth.2010.01.013","article-title":"High resolution melting analysis for gene scanning","volume":"50","author":"Erali","year":"2010","journal-title":"Methods"},{"key":"ref10","doi-asserted-by":"publisher","first-page":"484","DOI":"10.1016\/j.chom.2010.11.005","article-title":"Phosphorylation of immunity-related GTPases by a toxoplasma gondii-secreted kinase promotes macrophage survival and virulence","volume":"8","author":"Fentress","year":"2010","journal-title":"Cell Host Microbe"},{"key":"ref11","doi-asserted-by":"publisher","first-page":"842595","DOI":"10.3389\/fcimb.2022.842595","article-title":"Toxoplasma gondii genotyping: a closer look into Europe","volume":"12","author":"Fernandez-Escobar","year":"2022","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"371","DOI":"10.1007\/s11274-026-05107-5","article-title":"High-resolution melting (HRM)-based genotyping of toxoplasma gondii in meat products: comparative evaluation of ROP18, ROP5, and B1 gene markers","volume":"42","author":"Fotouhi-Ardakani","year":"2026","journal-title":"World J. Microbiol. Biotechnol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"e0227749","DOI":"10.1371\/journal.pone.0227749","article-title":"Molecular and biological analysis revealed genetic diversity and high virulence strain of toxoplasma gondii in Japan","volume":"15","author":"Fukumoto","year":"2020","journal-title":"PLoS One"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"1211","DOI":"10.3201\/eid1108.041354","article-title":"Coxiella burnetii genotyping","volume":"11","author":"Glazunova","year":"2005","journal-title":"Emerg. Infect. Dis."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"e0286430","DOI":"10.1371\/journal.pone.0286430","article-title":"Seroprevalence of toxoplasma gondii infection among patients of a tertiary hospital in Guangzhou, Guangdong province, PR China","volume":"18","author":"Guan","year":"2023","journal-title":"PLoS One"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"1561","DOI":"10.1093\/infdis\/172.6.1561","article-title":"Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease","volume":"172","author":"Howe","year":"1995","journal-title":"J. Infect. Dis."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"4278","DOI":"10.1038\/s41467-024-47625-6","article-title":"Far-east Asian toxoplasma isolates share ancestry with north and south\/central American recombinant lineages","volume":"15","author":"Ihara","year":"2024","journal-title":"Nat. Commun."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"e02280","DOI":"10.1128\/mBio.02280-14","article-title":"Toxoplasma gondii superinfection and virulence during secondary infection correlate with the exact ROP5\/ROP18 allelic combination","volume":"6","author":"Jensen","year":"2015","journal-title":"mBio"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"579","DOI":"10.1007\/s10096-017-3152-z","article-title":"Direct genetic characterization of toxoplasma gondii from clinical samples from Denmark: not only genotypes II and III","volume":"37","author":"Jokelainen","year":"2017","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"645","DOI":"10.1016\/j.ijpara.2011.01.005","article-title":"Genetic analyses of atypical toxoplasma gondii strains reveal a fourth clonal lineage in North America","volume":"41","author":"Khan","year":"2011","journal-title":"Int. J. Parasitol."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"14872","DOI":"10.1073\/pnas.0702356104","article-title":"Recent transcontinental sweep of toxoplasma gondii driven by a single monomorphic chromosome","volume":"104","author":"Khan","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"e1000404","DOI":"10.1371\/journal.pgen.1000404","article-title":"Selection at a single locus leads to widespread expansion of toxoplasma gondii lineages that are virulent in mice","volume":"5","author":"Khan","year":"2009","journal-title":"PLoS Genet."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"2980","DOI":"10.1093\/nar\/gki604","article-title":"Composite genome map and recombination parameters derived from three archetypal lineages of toxoplasma gondii","volume":"33","author":"Khan","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1186\/s13568-024-01728-x","article-title":"Rapid genotyping of toxoplasma gondii isolates via Nanopore-based multi-locus sequencing","volume":"14","author":"Koutsogiannis","year":"2024","journal-title":"AMB Express"},{"key":"ref25","doi-asserted-by":"publisher","first-page":"5586858","DOI":"10.1155\/japr\/5586858","article-title":"Evolution of toxoplasma gondii detection and genotyping methods","volume":"2026","author":"Kowalczyk","year":"2026","journal-title":"J. Parasitol. Res."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"172","DOI":"10.1186\/1746-6148-9-172","article-title":"Development and validation of TaqMan probe based real time PCR assays for the specific detection of genotype a and B small ruminant lentivirus strains","volume":"9","author":"Kuhar","year":"2013","journal-title":"BMC Vet. Res."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"655","DOI":"10.1093\/nar\/28.2.655","article-title":"3\u2032-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures","volume":"28","author":"Kutyavin","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"11423","DOI":"10.1073\/pnas.0601438103","article-title":"Globalization and the population structure of toxoplasma gondii","volume":"103","author":"Lehmann","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"892","DOI":"10.1111\/j.1574-6976.2009.00182.x","article-title":"Bacterial strain typing in the genomic era","volume":"33","author":"Li","year":"2009","journal-title":"FEMS Microbiol. Rev."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"292","DOI":"10.1186\/s13071-015-0902-6","article-title":"Diagnosis of toxoplasmosis and typing of toxoplasma gondii","volume":"8","author":"Liu","year":"2015","journal-title":"Parasit. Vectors"},{"key":"ref31","doi-asserted-by":"publisher","first-page":"10147","DOI":"10.1038\/ncomms10147","article-title":"Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic toxoplasma gondii genomes","volume":"7","author":"Lorenzi","year":"2016","journal-title":"Nat. Commun."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"623","DOI":"10.1177\/1087057110365900","article-title":"Application of high-resolution melting to large-scale, high-throughput SNP genotyping: a comparison with the TaqMan method","volume":"15","author":"Martino","year":"2010","journal-title":"J. Biomol. Screen."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"363","DOI":"10.1111\/j.1469-0691.2009.02809.x","article-title":"Evaluation of a new 5\u2032-nuclease real-time PCR assay targeting the toxoplasma gondii AF146527 genomic repeat","volume":"16","author":"Menotti","year":"2010","journal-title":"Clin. Microbiol. Infect."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"37","DOI":"10.30564\/vsr.v2i2.2673","article-title":"Genotyping of toxoplasma gondii in sheep and cattle meat using PCR-RFLP technique","volume":"2","author":"Rahdar","year":"2021","journal-title":"Vet. Sci. Res."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"1369","DOI":"10.1128\/JCM.02379-16","article-title":"Evaluation of toxoplasma ELITe MGB real-time PCR assay for diagnosis of toxoplasmosis","volume":"55","author":"Robert-Gangneux","year":"2017","journal-title":"J. Clin. Microbiol."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"264","DOI":"10.1128\/CMR.05013-11","article-title":"Epidemiology of and diagnostic strategies for toxoplasmosis","volume":"25","author":"Robert-Gangneux","year":"2012","journal-title":"Clin. Microbiol. Rev."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"1780","DOI":"10.1126\/science.1133690","article-title":"Polymorphic secreted kinases are key virulence factors in toxoplasmosis","volume":"314","author":"Saeij","year":"2006","journal-title":"Science"},{"key":"ref38","doi-asserted-by":"publisher","first-page":"108971","DOI":"10.1016\/j.exppara.2025.108971","article-title":"Exploring global trends in human toxoplasmosis seroprevalence by meta-analysis","volume":"275","author":"Sengupta","year":"2025","journal-title":"Exp. Parasitol."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"141","DOI":"10.1016\/j.ijpara.2015.10.005","article-title":"The ROP18 and ROP5 gene allele types are highly predictive of virulence in mice across globally distributed strains of toxoplasma gondii","volume":"46","author":"Shwab","year":"2016","journal-title":"Int. J. Parasitol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"82","DOI":"10.1038\/359082a0","article-title":"Virulent strains of toxoplasma gondii comprise a single clonal lineage","volume":"359","author":"Sibley","year":"1992","journal-title":"Nature"},{"key":"ref41","doi-asserted-by":"publisher","first-page":"10753","DOI":"10.1073\/pnas.172117099","article-title":"Identification of quantitative trait loci controlling acute virulence in toxoplasma gondii","volume":"99","author":"Su","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref42","doi-asserted-by":"publisher","first-page":"5844","DOI":"10.1073\/pnas.1203190109","article-title":"Globally diverse toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages","volume":"109","author":"Su","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1017\/S0031182009991065","article-title":"Moving towards an integrated approach to molecular detection and identification of toxoplasma gondii","volume":"137","author":"Su","year":"2010","journal-title":"Parasitology"},{"key":"ref44","doi-asserted-by":"publisher","first-page":"841","DOI":"10.1016\/j.ijpara.2006.03.003","article-title":"Genotyping of toxoplasma gondii by multilocus PCR-RFLP markers: a high resolution and simple method for identification of parasites","volume":"36","author":"Su","year":"2006","journal-title":"Int. J. Parasitol."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"e88","DOI":"10.4142\/jvs.2021.22.e88","article-title":"Toxoplasma gondii virulence prediction using hierarchical cluster analysis based on coding sequences (CDS) of sag1, gra7 and rop18","volume":"22","author":"Subekti","year":"2021","journal-title":"J. Vet. Sci."},{"key":"ref46","doi-asserted-by":"publisher","first-page":"101935","DOI":"10.1016\/j.parint.2019.101935","article-title":"A toxoplasma gondii strain isolated in Okinawa, Japan shows high virulence in Microminipigs","volume":"72","author":"Taniguchi","year":"2019","journal-title":"Parasitol. Int."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"1776","DOI":"10.1126\/science.1133643","article-title":"A secreted serine-threonine kinase determines virulence in the eukaryotic pathogen toxoplasma gondii","volume":"314","author":"Taylor","year":"2006","journal-title":"Science"},{"key":"ref48","doi-asserted-by":"publisher","first-page":"853","DOI":"10.1373\/49.6.853","article-title":"High-resolution genotyping by amplicon melting analysis using LCGreen","volume":"49","author":"Wittwer","year":"2003","journal-title":"Clin. Chem."},{"key":"ref49","doi-asserted-by":"publisher","first-page":"126293","DOI":"10.1016\/j.micres.2019.06.003","article-title":"Toxoplasma gondii secretory proteins and their role in invasion and pathogenesis","volume":"227","author":"Zhang","year":"2019","journal-title":"Microbiol. 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