【摘要】 目的 建立严重急性呼吸综合征冠状病毒2(severe acute respiratory symptom coronavirus 2,SARS-CoV-2)灭活疫苗（Vero细胞）中宿主细胞DNA残留量荧光定量PCR检测方法，并进行验证，以期更好地控制产品安全性。方法 通过磁珠法对SARS-CoV-2灭活疫苗（Vero细胞）原液进行DNA提取，采用探针型PCR对宿主细胞DNA残留量进行定量分析。对建立的方法进行线性范围、重复性、中间精密度、定量限、专属性、耐用性、准确度验证，并使用该方法对5批SARS-CoV-2灭活疫苗（Vero细胞）进行宿主细胞DNA残留量检测。结果 DNA标准曲线在300～0.003 pg/μL范围内线性良好（R~2均≥0.99）；重复性和中间精密度验证相对标准偏差（relative standard deviation,RSD）均<20%；定量限为0.001 pg/μL；样品稀释液与纯化液稀释液对检测无干扰；样品于53、55、57℃条件下孵育60 min及55℃条件下孵育56、60、64 min的检测结果无明显差异；准确度验证回收率在79%～83%,RSD <5%。用该方法检测SARS-CoV-2灭活疫苗（Vero细胞）原液中DNA残留量适应性良好。结论 成功建立了SARS-CoV-2灭活疫苗（Vero细胞）中宿主细胞DNA残留量荧光定量PCR检测方法，该方法线性范围、重复性、中间精密度、定量限、专属性、耐用性和准确度均符合可接受标准，适用于SARS-CoV-2灭活疫苗（Vero细胞）中宿主细胞DNA残留量的检测及质量控制。更多还原

【Abstract】 Objective To develop and verify a quantitative real-time PCR method for determination of the content of host cell DNA residues in severe acute respiratory symptom coronavirus 2(SARS-CoV-2) inactivated vaccine(Vero cells),in order to better control the safety of products.Methods DNA was extracted from inactivated SARS-CoV-2 vaccine(Vero cells) bulk by magnetic bead separation method,and the DNA residues of host cells were quantitatively analyzed by probetype PCR.The linear range,repeatability,intermediate precision,quantitative limit,specificity,durability and accuracy of the developed method were verified,and the host cell DNA re sidues of 5 batches of inactivated SARS-CoV-2 vaccine(Vero cells)were determined by this method.Results DNA standard curve showed good linearity in the range of 300～0.003 pg/μL(each R~2> 0.99);Relative standard deviations(RSD) of repeatability and intermediate precision verification were less than 20%;The quantitative limit was 0.001 pg/μL;Sample dilution and purified liquid dilution had no interference to detection;The results of 60 min incubation at 53,55,57 ℃ and 56,60,64 min incubation at 55 ℃ showed no significant difference;The recoveries of accuracy verification were 79%～83%,RSD <5%.This method had good adaptability in detecting DNA residues in the bulk of inactivated SARS-CoV-2 vaccine(Vero cells).Conclusion The quantitative realtime PCR method for determination of host cell DNA residues in inactivated SARS-CoV-2 vaccine(Vero cells) has been successfully developed,of which the linearity and range,repeatability,intermediate precision,quantitative limit,specificity,durability and accuracy meet the acceptable standards,and are suitable for the detection and quality control of host cell DNA residues in inactivated SARS-CoV-2 vaccine(Vero cells).更多还原