{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T04:23:11Z","timestamp":1781151791776,"version":"3.54.1"},"reference-count":53,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2020,8,12]],"date-time":"2020-08-12T00:00:00Z","timestamp":1597190400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["MicrobiologyOpen"],"published-print":{"date-parts":[[2020,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Upon acquiring two unique plasmids (pMT1 and pPCP1) and genome rearrangement during the evolution from <jats:italic>Yersinia pseudotuberculosis<\/jats:italic>, the plague causative agent <jats:italic>Y<\/jats:italic>.<jats:italic> pestis<\/jats:italic> is closely related to <jats:italic>Y<\/jats:italic>.<jats:italic> pseudotuberculosis<\/jats:italic> genetically but became highly virulent. We developed a pentaplex real\u2010time PCR assay that not only detects both <jats:italic>Yersinia<\/jats:italic> species but also differentiates <jats:italic>Y<\/jats:italic>.<jats:italic> pestis<\/jats:italic> strains regarding their plasmid profiles. The five targets used were <jats:italic>Y<\/jats:italic>.<jats:italic> pestis<\/jats:italic>\u2010specific <jats:italic>ypo2088<\/jats:italic>, <jats:italic>caf1<\/jats:italic>, and <jats:italic>pst<\/jats:italic> located on the chromosome, plasmids pMT1 and pPCP1, respectively; <jats:italic>Y<\/jats:italic>.<jats:italic> pseudotuberculosis<\/jats:italic>\u2010specific chromosomal gene <jats:italic>opgG<\/jats:italic>; and 18S ribosomal RNA gene as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1\u00a0fg to 100\u00a0fg, with <jats:italic>Y<\/jats:italic>.<jats:italic> pestis<\/jats:italic>\u2010specific <jats:italic>pst<\/jats:italic> as the most sensitive target. Using the assay, <jats:italic>Y<\/jats:italic>.<jats:italic> pestis<\/jats:italic> strains were differentiated 100% by their known plasmid profiles. Testing <jats:italic>Y<\/jats:italic>.<jats:italic> pestis<\/jats:italic> and <jats:italic>Y<\/jats:italic>.<jats:italic> pseudotuberculosis<\/jats:italic>\u2010spiked flea DNA showed there is no interference from flea DNA on the amplification of targeted genes. Finally, we applied the assay for testing 102 fleas collected from prairie dog burrows where prairie dog die\u2010off was reported months before flea collection. All flea DNA was amplified by 18S rRNA; no <jats:italic>Y<\/jats:italic>.<jats:italic> pseudotuberculosis<\/jats:italic> was detected; one flea was positive for all <jats:italic>Y<\/jats:italic>.<jats:italic> pestis<\/jats:italic>\u2010specific targets, confirming local <jats:italic>Y<\/jats:italic>.<jats:italic> pestis<\/jats:italic> transmission. Our results indicated the assay is sensitive and specific for the detection and differentiation of <jats:italic>Y<\/jats:italic>.<jats:italic> pestis<\/jats:italic> and <jats:italic>Y<\/jats:italic>.<jats:italic> pseudotuberculosis<\/jats:italic>. The assay can be used in field investigations for the rapid identification of the plague causative agent.<\/jats:p>","DOI":"10.1002\/mbo3.1105","type":"journal-article","created":{"date-parts":[[2020,8,12]],"date-time":"2020-08-12T08:10:53Z","timestamp":1597219853000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Pentaplex real\u2010time PCR for differential detection of <i>Yersinia pestis<\/i> and <i>Y<\/i>.<i> pseudotuberculosis<\/i> and application for testing fleas collected during plague epizootics"],"prefix":"10.1002","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7634-4051","authenticated-orcid":false,"given":"Ying","family":"Bai","sequence":"first","affiliation":[{"name":"Bacterial Disease Branch Division of Vector\u2010Borne Diseases Centers for Disease Control and Prevention Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vladimir","family":"Motin","sequence":"additional","affiliation":[{"name":"Department of Pathology Department of Microbiology &amp; Immunology The University of Texas Medical Branch at Galveston Galveston Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Russell E.","family":"Enscore","sequence":"additional","affiliation":[{"name":"Bacterial Disease Branch Division of Vector\u2010Borne Diseases Centers for Disease Control and Prevention Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lynn","family":"Osikowicz","sequence":"additional","affiliation":[{"name":"Bacterial Disease Branch Division of Vector\u2010Borne Diseases Centers for Disease Control and Prevention Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Rosales Rizzo","sequence":"additional","affiliation":[{"name":"Bacterial Disease Branch Division of Vector\u2010Borne Diseases Centers for Disease Control and Prevention Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrias","family":"Hojgaard","sequence":"additional","affiliation":[{"name":"Bacterial Disease Branch Division of Vector\u2010Borne Diseases Centers for Disease Control and Prevention Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Kosoy","sequence":"additional","affiliation":[{"name":"KB One Health LLC Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebecca J.","family":"Eisen","sequence":"additional","affiliation":[{"name":"Bacterial Disease Branch Division of Vector\u2010Borne Diseases Centers for Disease Control and Prevention Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2020,8,12]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.3201\/eid2402.160122"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.24.14043"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.3201\/eid2502.171974"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2016.00340"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1128\/mSphere.00402-17"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.65.5.1659-1668.1997"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.19.6.915-918.1970"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/0147-619X(81)90019-6"},{"key":"e_1_2_10_10_1","first-page":"89","article-title":"Plague around the world, 2010\u20132015","volume":"91","author":"Bertherat E.","year":"2016","journal-title":"Weekly Epidemiological Record"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0003532"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00248-003-1022-y"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.31.3.758-759.1993"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00507-18"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0404012101"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.1997.2731623.x"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0606831103"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.2000.62.552"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.1099\/0022-1317-49-3-261"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.31.2.839-841.1981"},{"key":"e_1_2_10_21_1","first-page":"306","article-title":"Sequencing of two Yersinia pestis IS elements, IS285 and IS100","volume":"13","author":"Filippov A. 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