{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T05:39:59Z","timestamp":1780465199942,"version":"3.54.1"},"reference-count":71,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2022,9,16]],"date-time":"2022-09-16T00:00:00Z","timestamp":1663286400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001711","name":"Swiss National Science Foundation","doi-asserted-by":"crossref","award":["310030_184662"],"award-info":[{"award-number":["310030_184662"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100017659","name":"Uniscientia Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100017659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (<jats:italic>mdhfr-ts<\/jats:italic>) drug selectable marker for pyrimethamine resistance in<jats:italic>Toxoplasma gondii<\/jats:italic>knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted<jats:italic>mdhfr-ts<\/jats:italic>and of the wild-type (WT)<jats:italic>dhfr-ts<\/jats:italic>(<jats:italic>wtdhfr-ts<\/jats:italic>) which is present as single copy gene in<jats:italic>Toxoplasma<\/jats:italic>and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the<jats:italic>dhfr-ts<\/jats:italic>fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of<jats:italic>dhfr-ts<\/jats:italic>copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp<jats:italic>dhfr-ts<\/jats:italic>fragment and the<jats:italic>T<\/jats:italic>.<jats:italic>gondii 529-bp<\/jats:italic>repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by<jats:italic>dhfr-ts<\/jats:italic>amplification is equal to the number of tachyzoites determined by amplification of the<jats:italic>Toxoplasma 529-bp<\/jats:italic>, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of<jats:italic>mdhfr-ts<\/jats:italic>, the calculated ratio is 2. We then applied both approaches to test<jats:italic>T<\/jats:italic>.<jats:italic>gondii<\/jats:italic>RH mutants in which the<jats:italic>major surface antigen<\/jats:italic>(SAG1) was disrupted through insertion of<jats:italic>mdhfr-ts<\/jats:italic>using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated<jats:italic>mdhfr-ts<\/jats:italic>. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of<jats:italic>T<\/jats:italic>.<jats:italic>gondii<\/jats:italic>KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of<jats:italic>mdhfr-ts<\/jats:italic>. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in<jats:italic>Toxoplasma<\/jats:italic>. TaqMan qPCR tools may contribute to more frequent use of WT<jats:italic>Toxoplasma<\/jats:italic>strains during functional genomics.<\/jats:p>","DOI":"10.1371\/journal.pone.0271011","type":"journal-article","created":{"date-parts":[[2022,9,16]],"date-time":"2022-09-16T17:37:43Z","timestamp":1663349863000},"page":"e0271011","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":4,"title":["Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9"],"prefix":"10.1371","volume":"17","author":[{"given":"Kai Pascal Alexander","family":"H\u00e4nggeli","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0622-2128","authenticated-orcid":true,"given":"Andrew","family":"Hemphill","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Norbert","family":"M\u00fcller","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1336-3671","authenticated-orcid":true,"given":"Bernd","family":"Schimanski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philipp","family":"Olias","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joachim","family":"M\u00fcller","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ghalia","family":"Boubaker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,9,16]]},"reference":[{"key":"pone.0271011.ref001","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1038\/s41579-021-00518-7","article-title":"Toxoplasma 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