摘要
目的 建立生物制品生产用细胞株逆转录酶活性SYBR Green玉实时定量PCR检测方法,并进行优化及初步验证。方法 以MS2噬菌体RNA模板及相应引物,建立一步检测逆转录酶扩增产物的SYBR Green玉实时荧光定量PCR方法,以梯度稀释的重组莫洛尼鼠白血病病毒（Moloney murine leukemia virus,MMLV）逆转录酶活性参考品绘制标准曲线,进行逆转录酶活性的定量。对反应的引物和2×RT-PCR Mix进行优化,并对建立的方法进行线性关系、灵敏度、精密度和准确度验证。结果1组引物（上游：5忆-CATAGGTCAAACCTCGTAGGAATG-3忆,下游：5忆-TCCTGCTC-AACTTCCTGTCGAG-3忆）更适合本方法;Promega公司生产的2×RT-PCR Mix具有更高的反应效率。逆转录酶活性在5.40×108～5.40×104pU/μl范围内定量标准曲线的线性关系良好,相关系数为0.999 7;该方法判断样品阴阳性的标准cut-off值为1.31×104pU/μl;检测CHO-1细胞株样品逆转录酶活性的试验内变异系数（CV）为0.80%,试验间CV值为16.4%;检测5株细胞样品逆转录酶活性的平均加标回收率为122.8%。结论 建立了生物制品生产用细胞株逆转录酶活性SYBR Green玉实时定量PCR检测方法,该方法能够准确地定量检测细胞株的逆转录酶活性。
Objective Objective To develop, optimize and preliminarily verify a SYBR Green I real-time PCR method for quantitative detection of reverse transcriptase activity in cell lines. Methods One-step SYBR Green I real-time PCR method for detection of amplified product of transeriptase was developed using MS2 bacteriophage RNA as a template and using the corresponding prhners. Standard curve was plotted by using serial dilutions of recombinant Moloney murine leukemia virus （MMLV） reverse transcriptase activity reference for quantitative detection of reverse transcriptase activity. The primers and 2 x RT-PCR Mix were optimized, and the developed method was verified for linearity, sensitivity, precision and accuracy. Results The primers in group 1 （upstream： 5＇-CATAGGTCAAACCTCGTAGGAATG-3＇, downstream： 5＇-TCCTGCTCAACTFCCTGTCGAG-3＇ ） were more suitable for the method. The 2 x RT-PCR Mix manufactured by Promega showed high reaction efficacy. The curve showed good linearity within a reverse transcriptase activity range of 5.40 × 10^8 5.40 × 10^4 pU/μl, with a correlation coefficient of 0. 999 7. The cut-off value of this method for sensitivity was 1.31 × 10^4 pU /μl. The CV values in intra- and inter-assays on reverse transcriptase aetivityin CHO-1 cells were 0. 80% and 16. 4% respectively. The mean spike recovery rate of reverse transcriptase activity in five cell strain was 122. 8%. Conclusion A SYBR Green I real-time PCR method for accurately quantitative detection of reverse transcriptase activity in cell lines for production of biologics was developed.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第8期1061-1065,共5页
Chinese Journal of Biologicals
基金
863计划项目(2012AA020805)
国家科技重大专项项目(2012ZX09304010)
关键词
细胞株
逆转录酶
活性
实时定量PCR
Cell line
Reverse transcriptase
Activity
Real-time quantitative PCR
