{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:15:34Z","timestamp":1760141734283,"version":"build-2065373602"},"reference-count":34,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T00:00:00Z","timestamp":1760054400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Vet. Sci."],"abstract":"<jats:p>Ensuring the safety and efficacy of veterinary vaccines requires reliable methods for detecting microbial contamination, particularly from <jats:italic>Mycoplasma<\/jats:italic> species, which pose a significant risk in cell-culture-derived vaccines. In the Republic of Korea, polymerase chain reaction (PCR) is predominantly used for <jats:italic>Mycoplasma<\/jats:italic> testing due to its faster turnaround compared to culture-based methods. However, in combination with vaccines containing <jats:italic>Erysipelothrix rhusiopathiae<\/jats:italic> and classical swine fever virus, PCR is rendered ineffective because of cross-reactivity between <jats:italic>Mycoplasma<\/jats:italic> universal primers and <jats:italic>E. rhusiopathiae<\/jats:italic>, resulting in non-specific amplification. This limitation necessitates reliance on the labor-intensive culture method, underscoring the need for more accurate and efficient alternatives. This study aimed to develop and validate next-generation sequencing (NGS)-based methods for detecting <jats:italic>Mycoplasma<\/jats:italic> contamination in veterinary vaccines and to compare their performance with that of PCR. Five species, including <jats:italic>Acholeplasma laidlawii<\/jats:italic> (genus <jats:italic>Acholeplasma<\/jats:italic>) and four <jats:italic>Mycoplasma<\/jats:italic> species\u2014<jats:italic>Mycoplasma fermentans<\/jats:italic>, <jats:italic>Mycoplasma orale<\/jats:italic>, <jats:italic>Mycoplasma hyorhinis<\/jats:italic>, and <jats:italic>Mycoplasma synoviae<\/jats:italic>\u2013were spiked into samples containing <jats:italic>E. rhusiopathiae<\/jats:italic>, a common vaccine component. Two NGS-based approaches were evaluated: (1) a reference-mapping method incorporating two-step alignment and <jats:italic>de novo<\/jats:italic> assembly, and (2) a 16S rRNA-based metabarcoding analysis using DADA2 and Qiime2. The reference-mapping method effectively filtered non-specific reads and accurately reconstructed <jats:italic>Mycoplasma<\/jats:italic>-derived contigs, whereas the metabarcoding approach enabled taxonomic profiling with quantitative resolution. The detection limits of NGS-based methods were substantially lower than those of PCR, demonstrating improvements of up to 100-fold depending on the species. Notably, omission of the initial mapping step resulted in excessive non-specific contig formation, highlighting the importance of the dual-step reference-mapping strategy. Although metabarcoding provided valuable abundance data, it was more prone to non-specific hits due to limited read overlap. In conclusion, the reference-mapping method demonstrated superior sensitivity, specificity, and quantification compared to both conventional PCR and metabarcoding, supporting its use as a robust tool for vaccine quality control. Implementing NGS-based detection methods could significantly enhance the safety and effectiveness of veterinary vaccines, ultimately enhancing vaccine quality control.<\/jats:p>","DOI":"10.3389\/fvets.2025.1657098","type":"journal-article","created":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T04:20:25Z","timestamp":1760070025000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Application of next-generation sequencing for detecting Mycoplasma contamination in veterinary vaccines"],"prefix":"10.3389","volume":"12","author":[{"given":"Su-Min","family":"Go","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yeon-Kyeong","family":"Lee","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jin-Ju","family":"Nah","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hyun-Ok","family":"Gu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Il","family":"Jang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Min-Goo","family":"Seo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,10,10]]},"reference":[{"key":"ref1","year":"2017"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"216","DOI":"10.22159\/ajpcr.2017.v10i1.14930","article-title":"Study the effect of Mycoplasma contamination of eggs used in virus titration and efficacy of some live attenuated poultry viral vaccines","volume":"10","author":"Fathy","year":"2017","journal-title":"Asian J Pharm Clin Res"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"196","DOI":"10.1016\/j.tim.2013.01.003","article-title":"Mycoplasmas and their host: emerging and re-emerging minimal pathogens","volume":"21","author":"Citti","year":"2013","journal-title":"Trends Microbiol"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"423","DOI":"10.2217\/fmb.11.18","article-title":"Mycoplasma pneumoniae: susceptibility and resistance to antibiotics","volume":"6","author":"B\u00e9b\u00e9ar","year":"2011","journal-title":"Future Microbiol"},{"key":"ref5","first-page":"210","article-title":"Mycoplasma","volume-title":"European Pharmacopoeia","year":"2023"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"109","DOI":"10.2353\/jmoldx.2010.090035","article-title":"The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5\u2032 nuclease assay","volume":"12","author":"Stadhouders","year":"2010","journal-title":"J Mol Diagn"},{"key":"ref7","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1385\/0-89603-525-5:145","article-title":"Characterization of mycoplasmas by PCR and sequence analysis with universal 16S rDNA primers","volume-title":"Mycoplasma protocols","author":"Johansson","year":"1998"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"1015","DOI":"10.1128\/cmr.00016-17","article-title":"Whole-genome sequencing of bacterial pathogens: the future of nosocomial outbreak analysis","volume":"30","author":"Quainoo","year":"2017","journal-title":"Clin Microbiol Rev"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"601","DOI":"10.1038\/nrg3226","article-title":"Transforming clinical microbiology with bacterial genome sequencing","volume":"13","author":"Didelot","year":"2012","journal-title":"Nat Rev Genet"},{"key":"ref10","volume-title":"VICH GL34: Biologicals: Testing for the detection of Mycoplasma contamination","year":"2014"},{"key":"ref11","first-page":"257","article-title":"Rapid and sensitive PCR method for identification of Mycoplasma species in tissue culture","author":"Wong-Lee","year":"1993","journal-title":"Diagnostic Molecular Microbiology: Principles and Applications"},{"key":"ref12","volume-title":"BBMap: A fast, accurate, splice-aware aligner. 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