{"title":"开发和验证在Vero细胞生产的狂犬病疫苗中检测残留宿主细胞DNA的qPCR方法。","authors":"Danhua Zhao, Weiying Zong, Wanxin Wu, Yuhua Li, Zongsong Wu, Zhixing Yang, Shouchun Cao","doi":"10.3389/fbioe.2025.1611428","DOIUrl":null,"url":null,"abstract":"<p><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 \"172bp\" 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 \"172bp\" 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.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"13 ","pages":"1611428"},"PeriodicalIF":4.8000,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12536431/pdf/","citationCount":"0","resultStr":"{\"title\":\"Development and validation of a qPCR assay for the detection of residual host cell DNA in rabies vaccines produced in Vero cells.\",\"authors\":\"Danhua Zhao, Weiying Zong, Wanxin Wu, Yuhua Li, Zongsong Wu, Zhixing Yang, Shouchun Cao\",\"doi\":\"10.3389/fbioe.2025.1611428\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><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 \\\"172bp\\\" 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 \\\"172bp\\\" 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.</p>\",\"PeriodicalId\":12444,\"journal\":{\"name\":\"Frontiers in Bioengineering and Biotechnology\",\"volume\":\"13 \",\"pages\":\"1611428\"},\"PeriodicalIF\":4.8000,\"publicationDate\":\"2025-10-06\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12536431/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Frontiers in Bioengineering and Biotechnology\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.3389/fbioe.2025.1611428\",\"RegionNum\":3,\"RegionCategory\":\"工程技术\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q1\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Bioengineering and Biotechnology","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.3389/fbioe.2025.1611428","RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/1/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Development and validation of a qPCR assay for the detection of residual host cell DNA in rabies vaccines produced in Vero cells.
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 "172bp" 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 "172bp" 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.
期刊介绍：
The translation of new discoveries in medicine to clinical routine has never been easy. During the second half of the last century, thanks to the progress in chemistry, biochemistry and pharmacology, we have seen the development and the application of a large number of drugs and devices aimed at the treatment of symptoms, blocking unwanted pathways and, in the case of infectious diseases, fighting the micro-organisms responsible. However, we are facing, today, a dramatic change in the therapeutic approach to pathologies and diseases. Indeed, the challenge of the present and the next decade is to fully restore the physiological status of the diseased organism and to completely regenerate tissue and organs when they are so seriously affected that treatments cannot be limited to the repression of symptoms or to the repair of damage. This is being made possible thanks to the major developments made in basic cell and molecular biology, including stem cell science, growth factor delivery, gene isolation and transfection, the advances in bioengineering and nanotechnology, including development of new biomaterials, biofabrication technologies and use of bioreactors, and the big improvements in diagnostic tools and imaging of cells, tissues and organs.
In today`s world, an enhancement of communication between multidisciplinary experts, together with the promotion of joint projects and close collaborations among scientists, engineers, industry people, regulatory agencies and physicians are absolute requirements for the success of any attempt to develop and clinically apply a new biological therapy or an innovative device involving the collective use of biomaterials, cells and/or bioactive molecules. “Frontiers in Bioengineering and Biotechnology” aspires to be a forum for all people involved in the process by bridging the gap too often existing between a discovery in the basic sciences and its clinical application.
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