{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,17]],"date-time":"2026-01-17T20:16:19Z","timestamp":1768680979455,"version":"3.49.0"},"reference-count":15,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2022,12,19]],"date-time":"2022-12-19T00:00:00Z","timestamp":1671408000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100012895","name":"Zoetis","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100012895","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003130","name":"Fonds Wetenschappelijk Onderzoek","doi-asserted-by":"publisher","award":["12W8919N"],"award-info":[{"award-number":["12W8919N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Avian pathogenic <jats:italic>Escherichia coli<\/jats:italic> (APEC) can cause colibacillosis in poultry, characterised by localised or systemic infections. Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated <jats:italic>Escherichia coli<\/jats:italic> O78 <jats:italic>aroA<\/jats:italic> gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the <jats:italic>aroA<\/jats:italic> gene, as well as the wild type <jats:italic>E<\/jats:italic>. <jats:italic>coli<\/jats:italic> strains by targeting the <jats:italic>xanQ<\/jats:italic> gene. The specificity of this PCR was evaluated using 123 <jats:italic>E<\/jats:italic>. <jats:italic>coli<\/jats:italic> isolates, form which 5 WT <jats:italic>aroA<\/jats:italic> auxotrophic strains (WT strains with a natural <jats:italic>aroA<\/jats:italic> deficiency), as well as 7 non-<jats:italic>Escherichia<\/jats:italic> isolates. The PCR showed 100% sensitivity of the <jats:italic>xanQ<\/jats:italic> primers for <jats:italic>E<\/jats:italic>. <jats:italic>coli<\/jats:italic> detection and 100% sensitivity of the \u0394<jats:italic>aroA<\/jats:italic> primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different <jats:italic>E<\/jats:italic>. <jats:italic>coli<\/jats:italic> strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*10<jats:sup>3<\/jats:sup> copies\/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total <jats:italic>E<\/jats:italic>. <jats:italic>coli<\/jats:italic> load in intestinal digesta from both vaccinated and non-vaccinated birds. <jats:italic>E<\/jats:italic>. <jats:italic>coli<\/jats:italic> could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. The duplex qPCR is helpful in monitoring colonization and shedding of the vaccine strain.<\/jats:p>","DOI":"10.1371\/journal.pone.0278949","type":"journal-article","created":{"date-parts":[[2022,12,19]],"date-time":"2022-12-19T18:30:31Z","timestamp":1671474631000},"page":"e0278949","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development of a duplex qPCR for the differentiation of a live attenuated\u00a0Escherichia coli\u00a0aroA mutant vaccine strain from field isolates in chickens"],"prefix":"10.1371","volume":"17","author":[{"given":"Kirsten","family":"Leurs","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6476-5932","authenticated-orcid":true,"given":"Evy","family":"Goossens","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4238-4658","authenticated-orcid":true,"given":"Henrik","family":"Christensen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8520-0834","authenticated-orcid":true,"given":"Jacques 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