{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,14]],"date-time":"2026-05-14T15:59:36Z","timestamp":1778774376768,"version":"3.51.4"},"reference-count":24,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T00:00:00Z","timestamp":1759708800000},"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. Bioeng. Biotechnol."],"abstract":"<jats:p>Residual host cell DNA in biological products, including rabies vaccines, poses potential health risks such as tumorigenesis and infectivity. Regulatory authorities have set limits for residual DNA levels to ensure product safety. Among various detection methods for residual DNA, quantitative PCR (qPCR) is recognized for its high sensitivity and efficiency. This study developed and validated a qPCR assay for detecting residual Vero DNA in rabies vaccines produced in Vero cells. The assay targeted two highly repetitive Vero genomic DNA sequences: the \u201c172bp\u201d sequence and the Alu repetitive sequence. The method was optimized and validated for linearity, range, quantitation limit, detection limit and specificity, etc. The qPCR assay for the \u201c172bp\u201d sequence exhibited excellent linearity, with a quantification limit of 0.03pg\/reaction and a detection limit of 0.003pg\/reaction. The relative standard deviation (RSD) across samples ranged from 12.4% to 18.3%, and the recovery rate was between 87.7% and 98.5%. No cross-reactivity was observed with common bacterial and cell strains, indicating a high specificity of the assay. These findings suggest that the qPCR method is a reliable approach for quantifying residual Vero DNA in pharmaceuticals and for regulatory compliance monitoring. The assay method has been adopted by local vaccine manufacturers, and has been included in the Chinese Pharmacopoeia, thus will help to enhance vaccine quality and safety.<\/jats:p>","DOI":"10.3389\/fbioe.2025.1611428","type":"journal-article","created":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T12:38:11Z","timestamp":1759754291000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Development and validation of a qPCR assay for the detection of residual host cell DNA in rabies vaccines produced in Vero cells"],"prefix":"10.3389","volume":"13","author":[{"given":"Danhua","family":"Zhao","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weiying","family":"Zong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wanxin","family":"Wu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuhua","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zongsong","family":"Wu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhixing","family":"Yang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shouchun","family":"Cao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,10,6]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"139","DOI":"10.1016\/j.biologicals.2016.03.002","article-title":"Universal realtime PCR assay for quantitation and size evaluation of residual cell DNA in human viral vaccines","volume":"44","author":"Andr\u00e9","year":"2016","journal-title":"Biologicals"},{"key":"B2","doi-asserted-by":"publisher","first-page":"413","DOI":"10.4161\/hv.22699","article-title":"Development of a vero cell DNA reference standard for residual DNA measurement in China","volume":"9","author":"Cao","year":"2013","journal-title":"Hum. Vaccines Immunother."},{"key":"B3","volume-title":"Pharmacopoeia of the people\u2019s Republic of China","year":"2015"},{"key":"B4","volume-title":"Pharmacopoeia of the people\u2019s Republic of China","year":"2020"},{"key":"B5","article-title":"European pharmacopeia","volume-title":"Characterisation of residual host-cell DNA","year":"2025"},{"key":"B6","volume-title":"Guidance for industry characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications","year":"2010"},{"key":"B7","doi-asserted-by":"publisher","first-page":"13202","DOI":"10.1038\/s41598-017-13402-3","article-title":"Highly sensitive and specific Alu-based quantification of human cells among rodent cells","volume":"7","author":"Funakoshi","year":"2017","journal-title":"Sci. Rep."},{"key":"B8","doi-asserted-by":"publisher","first-page":"5553","DOI":"10.1093\/nar\/9.21.5553","article-title":"Interspersed repeated sequences in the African green monkey genome that are homologous to the human alu family","volume":"9","author":"Grimaldi","year":"1981","journal-title":"Nucleic Acids Res."},{"key":"B9","first-page":"1892","article-title":"The probe selection in vero cells DNA residue detection by spot hybridization","volume":"28","author":"Li","year":"2008","journal-title":"Chin. J. Pharm. Anal."},{"key":"B10","doi-asserted-by":"publisher","first-page":"259","DOI":"10.1016\/j.biologicals.2009.04.002","article-title":"Quantitative determination of the infectivity of the proviral DNA of a retrovirus in vitro: evaluation of methods for DNA inactivation","volume":"37","author":"Li","year":"2009","journal-title":"Biologicals"},{"key":"B11","doi-asserted-by":"publisher","first-page":"e108926","DOI":"10.1371\/journal.pone.0108926","article-title":"A mouse strain defective in both T cells and NK cells has enhanced sensitivity to tumor induction by plasmid DNA expressing both activated H-Ras and c-Myc","volume":"9","author":"Li","year":"2014","journal-title":"PLoS One"},{"key":"B12","doi-asserted-by":"publisher","first-page":"1379","DOI":"10.3390\/vaccines12121379","article-title":"Evaluation of the efficacy of the vaccine production process in removing residual host cell DNA from the vero cell rabies vaccine","volume":"12","author":"Li","year":"2024","journal-title":"Vaccines"},{"key":"B13","doi-asserted-by":"publisher","first-page":"673","DOI":"10.1093\/dnares\/dsu029","article-title":"The genome landscape of the African green monkey kidney-derived vero cell line","volume":"21","author":"Osada","year":"2014","journal-title":"DNA Res."},{"key":"B14","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1016\/j.jpba.2014.07.032","article-title":"Direct real-time quantitative PCR for measurement of host-cell residual DNA in therapeutic proteins","volume":"100","author":"Peper","year":"2014","journal-title":"J. Pharm. Biomed. Anal."},{"key":"B15","doi-asserted-by":"publisher","first-page":"5506","DOI":"10.1016\/s0021-9258(18)50624-4","article-title":"Arrangement of a highly repeated DNA sequence in the genome and chromatin of the African green monkey","volume":"254","author":"Singer","year":"1979","journal-title":"J. Biol. Chem."},{"key":"B16","article-title":"United States pharmacopeia. Chapter 509: residual DNA testing","year":"2025"},{"key":"B17","volume-title":"The United States pharmacopeial convention. Chapter 1130: nucleic acid-based techniques - approaches for detecting trace nucleic acids (residual DNA testing)","year":"2025"},{"key":"B18","doi-asserted-by":"publisher","first-page":"101674","DOI":"10.1016\/j.mcp.2020.101674","article-title":"Design and development of a simple method for the detection and quantification of residual host cell DNA in recombinant rotavirus vaccine","volume":"55","author":"Varnamkhasti","year":"2021","journal-title":"Mol. Cell. Probes."},{"key":"B19","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1016\/j.jviromet.2019.01.001","article-title":"Comparative analysis of the performance of residual host cell DNA assays for viral vaccines produced in vero cells","volume":"268","author":"Vernay","year":"2019","journal-title":"J. Virol. Methods"},{"key":"B20","article-title":"Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks","volume":"3","year":"2025","journal-title":"Annex"},{"key":"B21","doi-asserted-by":"publisher","first-page":"1597","DOI":"10.1586\/erv.11.140","article-title":"From brain passage to cell adaptation: the road of human rabies vaccine development","volume":"10","author":"Wu","year":"2011","journal-title":"Expert Rev. Vaccines"},{"key":"B22","first-page":"1201","article-title":"The research for the SV40 by means of tissue culture technique","volume":"21","author":"Yasumura","year":"1963","journal-title":"Nippon. Rinsho"},{"key":"B23","doi-asserted-by":"publisher","first-page":"145","DOI":"10.1016\/j.jpba.2014.07.037","article-title":"Development and qualification of a high sensitivity, high throughput Q-PCR assay for quantitation of residual host cell DNA in purification process intermediate and drug substance samples","volume":"100","author":"Zhang","year":"2014","journal-title":"J. Pharm. Biomed. Anal."},{"key":"B24","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1186\/s12575-019-0105-1","article-title":"Development and validation of quantitative real-time PCR for the detection of residual CHO host cell DNA and optimization of sample pretreatment method in biopharmaceutical products","volume":"21","author":"Zheng","year":"2019","journal-title":"Biol. Proced. Online"}],"container-title":["Frontiers in Bioengineering and Biotechnology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fbioe.2025.1611428\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,7]],"date-time":"2025-10-07T15:35:23Z","timestamp":1759851323000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fbioe.2025.1611428\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,6]]},"references-count":24,"alternative-id":["10.3389\/fbioe.2025.1611428"],"URL":"https:\/\/doi.org\/10.3389\/fbioe.2025.1611428","relation":{},"ISSN":["2296-4185"],"issn-type":[{"value":"2296-4185","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,6]]},"article-number":"1611428"}}