摘要
动物源性生物材料的残留DNA定量检测是产品脱细胞处理过程是否彻底,以及免疫原性风险是否得到有效控制的重要产品技术指标之一。目前,国际上还没有针对这类材料的残留DNA检测方法。本研究设计了动物源性生物材料的残留DNA定量检测三步法,即固体生物材料的蛋白酶K消化,DNA纯化和荧光染色法DNA测定。在整个实验过程中增加了回收率实验,经过回收率曲线方程校正后得到最终检测结果。实验过程中的磁珠法DNA纯化步骤的优化设计保证了较好的回收率,同时满足了准确性和精密度。该实验方法经验证,其检测灵敏度达到6.25ng/每份样品,回收率样品DNA含量在3.125～100ng以及25～400ng范围内线性良好,其回收率曲线R2>0.99。此方法保证了生物材料中微量或痕量DNA检测的科学性和可信性。
Quantification of residual DNA in animal-derived biological scaffold materials is one of technical specifica- tions for evaluating decellularization process and immunotoxieity risk. Up to now, there have been no standard meth- ods available for quantification of residues DNA in animal-derived biological scaffold materials. In this study, a three- step method, including proteinase K digestion, DNA purification and determination of DNA using fluorescence as- say, was designed for residual DNA quantification. A parallel recovery experiment of standard DNA using the same protocol to test article determination was used for adjusting final results of residuul DNA amount. DNA purification based on magnetic beads enabled the experiments to get high accuracy and repeatability. The validation experiment showed that the three-step method had high sensitivity up to 6.25ng of DNA per sample with good linearity （recover- y curve R2〉0.99） in the concentration range of 3. 125-100ng, and 25-400ng per sample. This method is useful for determining micro or trace amount DNA remained in the biomaterials.
出处
《生物医学工程学杂志》
EI
CAS
CSCD
北大核心
2012年第3期479-485,共7页
Journal of Biomedical Engineering
基金
中国食品药品检定研究院中青年研究发展基金课题资助(2011C2)
