用于鉴别猴源细胞及其交叉污染短串联重复序列图谱分析方法的建立
|
摘要
目的建立用于鉴别猴源细胞及其交叉污染的短串联重复序列(Short Tandem Repeat,STR)图谱分析方法。方法选择可用于猴源细胞鉴别的10个STR位点,采用PCR-毛细管电泳分析方法分别检测猴源、人源、鼠源细胞及猴源细胞交叉污染模型细胞中的10个STR位点,并验证方法的特异性及稳定性。同时对5家不同受检单位生产用的Vero细胞进行STR图谱分析。结果 STR图谱分析法可为每一个独立的猴源细胞系提供独特的STR图谱,并能有效判断猴源细胞与其他细胞间的交叉污染,且特异性及稳定性较高。采用该方法检测的5株不同受检单位生产用的Vero细胞均未发生污染。结论建立的STR图谱分析方法具有良好的特异性和稳定性,可用于不同猴源细胞系的鉴别和有效判断相关细胞间交叉污染。
Objective To establish a short tandem repeat(STR) profiling-based method for cell identification and evaluation of cross-contamination of monkey cell lines. Methods Ten individual STR loci were collected with their informativity being assessed to indicate the genetic polymorphism of a given chromosomal locus. PCR-capillary electrophoresis was performed to examine the 10-loci STR profiling in the cell lines derived from human being,monkey,mouse,and the cells mixed from different lines,and verified for specificity and stability. Five monkey cell lines used for production of biologics were analyzed by this method. Results The use of the newly established method generated unique STR profiling for each monkey cell line,by which the cross-contamination of monkey cell line and other cell lines was effectively judged. Conclusion The established STR profiling showed high specificity,sensitivity and stability,which might be used for identification and evaluation of cross-contamination of monkey cell lines.
Objective To establish a short tandem repeat(STR) profiling-based method for cell identification and evaluation of cross-contamination of monkey cell lines. Methods Ten individual STR loci were collected with their informativity being assessed to indicate the genetic polymorphism of a given chromosomal locus. PCR-capillary electrophoresis was performed to examine the 10-loci STR profiling in the cell lines derived from human being,monkey,mouse,and the cells mixed from different lines,and verified for specificity and stability. Five monkey cell lines used for production of biologics were analyzed by this method. Results The use of the newly established method generated unique STR profiling for each monkey cell line,by which the cross-contamination of monkey cell line and other cell lines was effectively judged. Conclusion The established STR profiling showed high specificity,sensitivity and stability,which might be used for identification and evaluation of cross-contamination of monkey cell lines.
引文
[1]FDA.Guidance for Industry:Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications[J].Fed Regist,2010,75(42):9905.
[2]NA T,YUAN B Z.A cytochrome b based PCR method to rapidly determine cell species and inter-speices cross-contamination[J].Chin J Pharm Anal,2014,34(11):2054-2059.(in Chinese)纳涛,袁宝珠.基于细胞色素b序列差异的细胞种属鉴别及种属间细胞交叉污染的快速检测方法[J].药物分析杂志,2014,34(11):2054-2059.
[3]Chinese Pharmacopoeia Commission.Pharmacopoeia of People's Repubic of China(VolⅢ)[S].Beijing:China Med Sci Press,2015:21.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2015:21.
[4]REID Y,STORTS D,RISS T,et al.Authentication of human cell lines by STR DNA profiling analysis[S].Assay Guidance Manual,2012.
[5]WAHLS W P,WALLACE L J,MOORE P D.Hypervariable minisatellite DNA is a hotspot for homologous recombination in human cells[J].Cell,1990,60(1):95-103.
[6]YASUMURA Y,KAWAKITA Y.Studies on SV40 in tissue culture:preliminary step for cancer research in vitro(in Japanese)[J].Nihon Rinsho,1963,21:1201-1215.
[7]ALMEIDA J L,HILL C R,COLE K D.Authentication of African green monkey cell lines using human short tandem repeat markers[J].BMC Biotechnol,2011,11:102.doi:10.1186/1472-6750-11-102.
[8]WU H Z,LI J H,ZHAO J Y.Specific detection method to distinguish Tibetan macaca from human and common animal samples[J].J Biol,2007,24(1):18-21.(in Chinese)吴华彰,李进华,赵建元.短尾猴样品中常见哺乳动物及人源污染的特异性研究[J].生物学杂志,2007,24(1):18-21.
[9]MILANESI E,AJMONE-MARSAN P,BIGNOTTI E,et al.Molecular detection of cell line cross-contaminations using amplified fragment length polymorphism DNA fingerprinting technology[J].In Vitro Cell Dev Biol Anim,2003,39(3-4):124-130.
[10]HIROSHIGE Y,OHTAKI H,YOSHIMOTO T,et al.Species speci cities among primates probed with commercially available uorescence-based multiplex PCR typing kits[J].Legal Med(Tokyo),2015,17(5):326-333.
[2]NA T,YUAN B Z.A cytochrome b based PCR method to rapidly determine cell species and inter-speices cross-contamination[J].Chin J Pharm Anal,2014,34(11):2054-2059.(in Chinese)纳涛,袁宝珠.基于细胞色素b序列差异的细胞种属鉴别及种属间细胞交叉污染的快速检测方法[J].药物分析杂志,2014,34(11):2054-2059.
[3]Chinese Pharmacopoeia Commission.Pharmacopoeia of People's Repubic of China(VolⅢ)[S].Beijing:China Med Sci Press,2015:21.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2015:21.
[4]REID Y,STORTS D,RISS T,et al.Authentication of human cell lines by STR DNA profiling analysis[S].Assay Guidance Manual,2012.
[5]WAHLS W P,WALLACE L J,MOORE P D.Hypervariable minisatellite DNA is a hotspot for homologous recombination in human cells[J].Cell,1990,60(1):95-103.
[6]YASUMURA Y,KAWAKITA Y.Studies on SV40 in tissue culture:preliminary step for cancer research in vitro(in Japanese)[J].Nihon Rinsho,1963,21:1201-1215.
[7]ALMEIDA J L,HILL C R,COLE K D.Authentication of African green monkey cell lines using human short tandem repeat markers[J].BMC Biotechnol,2011,11:102.doi:10.1186/1472-6750-11-102.
[8]WU H Z,LI J H,ZHAO J Y.Specific detection method to distinguish Tibetan macaca from human and common animal samples[J].J Biol,2007,24(1):18-21.(in Chinese)吴华彰,李进华,赵建元.短尾猴样品中常见哺乳动物及人源污染的特异性研究[J].生物学杂志,2007,24(1):18-21.
[9]MILANESI E,AJMONE-MARSAN P,BIGNOTTI E,et al.Molecular detection of cell line cross-contaminations using amplified fragment length polymorphism DNA fingerprinting technology[J].In Vitro Cell Dev Biol Anim,2003,39(3-4):124-130.
[10]HIROSHIGE Y,OHTAKI H,YOSHIMOTO T,et al.Species speci cities among primates probed with commercially available uorescence-based multiplex PCR typing kits[J].Legal Med(Tokyo),2015,17(5):326-333.