摘要
目的对Taq Man MGB探针实时定量PCR检测Vero细胞残余DNA的方法进行适用性验证及应用。方法对Taq Man MGB探针实时定量PCR检测Vero细胞残余DNA的方法进行特异性、灵敏度、精密性、准确性验证,并应用该方法检测3批次脊髓灰质炎病毒浓缩液、纯化液和原液中Vero细胞残余DNA含量,计算Vero细胞残余DNA去除率。结果 Taq Man MGB探针实时定量PCR能够特异性扩增Vero细胞基因组DNA长串连重复序列;DNA浓度在10-1~10-6 ng/μl范围内标准曲线线性良好,相关系数为0.996,扩增效率为93.54%;检测灵敏度为10-6 ng/μl,高于斑点杂交法;检测高（10-2 ng/μl）、中（10-4 ng/μl）、低（10-6 ng/μl）浓度阳性对照DNA模板的试验内变异系数（CV）为0.145%~3.110%,试验间CV为0.624%~2.359%;检测不同浓度阳性对照DNA的回收率在101.5%~109.4%之间,均在定量方法可接受的回收率范围内;在斑点杂交检测灵敏度范围内,同一样品采用Taq Man MGB探针实时定量PCR法与斑点杂交法半定量检测的结果吻合。3批次脊髓灰质炎病毒浓缩液纯化后可有效去除Vero细胞残余DNA,总去除率约为100%。结论 Taq Man探针实时定量PCR法特异性、精密性、准确性良好,灵敏度高,可作为斑点杂交法的替代方法,用于实验室内部的质量控制。
Objective To validate the suitability and apply Taq Man MGB probe real-time quantitative PCR for the detection of residual DNA in Vero cells. Methods The Taq Man MGB probe real-time quantitative PCR was validated for specificity,sensitivity,precision and accuracy,and used for detection of residual Vero cell DNA contents in three batches of concentrated,purified and bulk poliovirus,based on which the removal rate of residual Vero cell DNA was calculated. Results The Vero cell genomic DNA long tandem repeats were amplified by the developed Taq Man MGB probe real-time quantitative PCR specifically. Standard curve showed good linearity within a DNA concentration range of10-1~ 10-6ng / μl,with a correlation coefficient（R2 value） of 0. 996 and an amplification efficacy of 93. 54%. The sensitivity of the developed method was 10-6ng / μl,which was higher than that of dot blot. The coefficients of variation（CVs）of determination results of positive control DNA templates at high（10-2 ng / μl）,moderate（10-4 ng / μl）and low（10-6 ng / μl）concentrations in intra-assay were 0. 145% ~ 3. 110%,while those in inter-assay were 0. 624% ~ 2.359%,respectively. The recovery rates of positive control DNA at various concentrations were 101. 5% ~ 109. 4%,which were within the receptible range. The result of residual Vero cell DNA concentration detected by Taq Man MGB probe real-time quantitative PCR was in line with that by dot blot within the sensitivity range. The total removal rate of residual Vero cell DNA in three batches of inactivated polio vaccine after purification was nearly 100%. Conclusion Taq Man probe realtime quantitative PCR assay showed high specificity,precision,accuracy and sensitivity,which may be used for in-house quality control of residual Vero cell DNA instead of dot blot hybridization.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第11期1443-1447,共5页
Chinese Journal of Biologicals
基金
科技部863计划(2012AA02A401)
国药新产品基金(2013SW22)
重大新药创制(2013ZX09402302)
北京市科委基金资助(Z131100006513007)
