{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T02:26:49Z","timestamp":1790389609419,"version":"4.1.0"},"reference-count":35,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2025,8,14]],"date-time":"2025-08-14T00:00:00Z","timestamp":1755129600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32302898"],"award-info":[{"award-number":["32302898"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"the Earmarked Fund for Doctoral Research Start-Up Fund of Henan University of Science and Technology","award":["13480105"],"award-info":[{"award-number":["13480105"]}]}],"content-domain":{"domain":["www.plosntds.org"],"crossmark-restriction":false},"short-container-title":["PLoS Negl Trop Dis"],"abstract":"<jats:p>\n                    <jats:italic>\n                      <jats:italic>Balantioides coli<\/jats:italic>\n                    <\/jats:italic>\n                    is a zoonotic ciliated protozoan that infects humans and other mammals. Conventional and ITS-based genotyping approaches have limitations that hinder precise molecular epidemiological investigations. The objective of this study was to develop a new \u03b2-tubulin gene-based approach to enhance the detection and genotyping of\n                    <jats:italic>\n                      <jats:italic>B. coli<\/jats:italic>\n                    <\/jats:italic>\n                    . We performed single-cell isolation and whole-genome sequencing on two\n                    <jats:italic>\n                      <jats:italic>B. coli<\/jats:italic>\n                    <\/jats:italic>\n                    isolates from pigs and two from guinea pigs. We then used the \u03b2-tubulin gene sequences to design PCR primers for the new genotyping assay. We validated the assay using 56 ITS-confirmed\n                    <jats:italic>\n                      <jats:italic>B. coli<\/jats:italic>\n                    <\/jats:italic>\n                    -positive fecal DNA samples from pigs, cattle, sheep, and guinea pigs. Phylogenetic analyses were conducted using both \u03b2-tubulin and ITS sequences. The \u03b2-tubulin-based nested PCR assay exhibited 100% detection efficiency and greater specificity than ITS-based methods. Phylogenetic analysis of the \u03b2-tubulin gene sequences classified\n                    <jats:italic>\n                      <jats:italic>B. coli<\/jats:italic>\n                    <\/jats:italic>\n                    into three genotypes (I-III). Genotype III appears to be specific to guinea pigs. Genotypes I and II were found across multiple hosts, indicating potential cross-species transmission. Of the five full-length\n                    <jats:italic>\n                      <jats:italic>B. coli<\/jats:italic>\n                    <\/jats:italic>\n                    \u03b2-tubulin sequences obtained in this study, 264 polymorphic sites (19.8%) were identified, including both synonymous and non-synonymous mutations. Frequent recombination events within the \u03b2-tubulin locus were detected, indicating substantial genetic diversity. Therefore, the \u03b2-tubulin gene is a robust marker for genotyping and epidemiological studies of\n                    <jats:italic>\n                      <jats:italic>B. coli<\/jats:italic>\n                    <\/jats:italic>\n                    . The novel nested PCR assay overcomes the limitations of ITS-based methods and has produced data revealing previously unrecognized genetic diversity and host specificity patterns of\n                    <jats:italic>\n                      <jats:italic>B. coli<\/jats:italic>\n                    <\/jats:italic>\n                    .\n                  <\/jats:p>","DOI":"10.1371\/journal.pntd.0013426","type":"journal-article","created":{"date-parts":[[2025,8,14]],"date-time":"2025-08-14T17:58:34Z","timestamp":1755194314000},"page":"e0013426","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development and application of a novel beta-tubulin genotyping tool reveals host-specific transmission cluster in Balantioides 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