{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,4]],"date-time":"2026-05-04T03:40:50Z","timestamp":1777866050039,"version":"3.51.4"},"reference-count":25,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T00:00:00Z","timestamp":1777507200000},"content-version":"vor","delay-in-days":119,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,1,1]],"date-time":"2026-01-01T00:00:00Z","timestamp":1767225600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32202812"],"award-info":[{"award-number":["32202812"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Transboundary and Emerging Diseases"],"published-print":{"date-parts":[[2026,1]]},"abstract":"<jats:p>\n                    <jats:italic>Mycoplasma hyopneumoniae<\/jats:italic>\n                    (\n                    <jats:italic>M. hyopneumoniae<\/jats:italic>\n                    ), the etiological agent of porcine enzootic pneumonia, is one of the most prevalent and economically significant respiratory pathogens in the swine industry. Vaccination constitutes a primary strategy for controlling\n                    <jats:italic>M. hyopneumoniae<\/jats:italic>\n                    infections. Live\u2010attenuated vaccines confer protection against wild\u2010type\n                    <jats:italic>M. hyopneumoniae<\/jats:italic>\n                    through competitive exclusion of early lung colonization and induce multifaceted immune responses, including humoral, mucosal, and cellular immunity, thereby exhibiting superior protective efficacy. However, the lack of methods for differentiating live\u2010attenuated vaccine immunization from wild\u2010type infection has hindered the widespread application of live\u2010attenuated vaccines. In this study, we developed a quantitative real\u2010time polymerase chain reaction (qPCR) method based on the ribonuclease H2 (RNase H2) cleavage principle, which emits specific fluorescent signals exclusively when the probe is fully complementary to the template of the live\u2010attenuated vaccine strains. This qPCR method exhibited high specificity and sensitivity for the\n                    <jats:italic>M. hyopneumoniae<\/jats:italic>\n                    live\u2010attenuated vaccines (168\u2010L and RM48), with a lower detection limit of 10\u2009copies\/\u00b5L. Using this qPCR method, the bacterial shedding dynamics of the 168\u2010L vaccine strain in piglets were effectively monitored, with the first detection in the nasal cavity at 3 days postvaccination (dpv), and a peak detection rate of 100% between 10 and 17 dpv. This established assay can specifically monitor the respiratory colonization and shedding of the vaccine strain after\n                    <jats:italic>M. hyopneumoniae<\/jats:italic>\n                    live\u2010attenuated vaccine immunization, thereby providing crucial technical support for the clinical evaluation of immunization efficacy.\n                  <\/jats:p>","DOI":"10.1155\/tbed\/5823134","type":"journal-article","created":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T07:53:41Z","timestamp":1777535621000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A RNase H2\u2010Linked TaqMan\u2010MGB Quantitative Real\u2010Time PCR Assay for Differential Detection of\n                    <i>Mycoplasma hyopneumoniae<\/i>\n                    Live\u2010Attenuated Vaccine Strains"],"prefix":"10.1155","volume":"2026","author":[{"given":"Kangyao","family":"Peng","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanna","family":"Wei","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuzi","family":"Wu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ting","family":"Yuan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiahao","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ping","family":"Jiang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2033-8565","authenticated-orcid":false,"given":"Qingyun","family":"Xie","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9113-4706","authenticated-orcid":false,"given":"Zhixin","family":"Feng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2026,4,30]]},"reference":[{"key":"e_1_2_12_1_2","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1111\/tbed.12677","article-title":"Update on Mycoplasma hyopneumoniae Infections in Pigs: Knowledge Gaps for Improved Disease Control","volume":"65","author":"Maes D.","year":"2018","journal-title":"Transboundary and Emerging Diseases"},{"key":"e_1_2_12_2_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12917-021-03059-6"},{"key":"e_1_2_12_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetmic.2007.09.008"},{"key":"e_1_2_12_4_2","doi-asserted-by":"publisher","DOI":"10.1080\/21505594.2020.1842659"},{"key":"e_1_2_12_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.rvsc.2019.01.007"},{"key":"e_1_2_12_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2022.08.072"},{"key":"e_1_2_12_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.38.4.1390-1396.2000"},{"key":"e_1_2_12_8_2","doi-asserted-by":"publisher","DOI":"10.1002\/vms3.539"},{"key":"e_1_2_12_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetmic.2018.07.023"},{"key":"e_1_2_12_10_2","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1111\/jam.13770","article-title":"A New Multiplex Real-Time TaqMan PCR for Quantification of Mycoplasma hyopneumoniae, Flocculare: Exploratory Epidemiological Investigations to Research Mycoplasmal Association in Enzootic Pneumonia-Like Lesions in Slaughtered Pigs","volume":"125","author":"Fourour S.","year":"2018","journal-title":"Journal of Applied Microbiology"},{"key":"e_1_2_12_11_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2024.1469558"},{"key":"e_1_2_12_12_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12917-017-1194-1"},{"key":"e_1_2_12_13_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13071-020-3994-6"},{"key":"e_1_2_12_14_2","doi-asserted-by":"publisher","DOI":"10.3389\/fvets.2025.1619301"},{"key":"e_1_2_12_15_2","first-page":"1347","article-title":"[Accurate Titration of Mycoplasma Culture Measured by 50% Color Change Unit Assay]","volume":"53","author":"Shen Q.","year":"2013","journal-title":"Wei Sheng Wu Xue Bao (Acta Microbiologica 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