摘要
目的建立可定量检测门冬氨酸鸟氨酸原料药中毕赤酵母菌DNA残留量的实时荧光定量PCR方法。方法采用宿主细胞残留DNA样本前处理试剂盒(磁珠法)提取毕赤酵母菌DNA,利用毕赤酵母残留DNA检测试剂盒(PCR-荧光探针法)进行扩增反应,绘制标准曲线,建立毕赤酵母菌DNA残留量的Real-time PCR检测方法,并验证其准确性和精密性。结果毕赤酵母菌DNA质量浓度在0.03~300 pg·μL-(16)内线性良好(r>0.99),定量限为0.03 pg·μL-(16);应用该法对3批门冬氨酸鸟氨酸原料药进行测定,毕赤酵母菌DNA残留量均远低于每剂10 ng。结论该方法可用于门冬氨酸鸟氨酸原料药中毕赤酵母菌DNA残留量的定量测定。
OBJECTIVE To establish a Real-time PCR method for quantitative detection of the Pichia pastoris DNA residues in ornithine aspartate. METHODS Genomic DNA of Pichia pastoris was extracted by using host cell residual DNA sample pretreatment kit based on magnetic beads. The reaction system and condition for Real-time PCR were determined, and a standard curve was plotted based on residual DNA kit for detection of Pichia pastoris by fluorescent probe. The Real-time PCR method was verified for accuracy and precision. RESULTS The calibration curve was linear in the range of 0.03-300 pg·μL^-1, with r0.99, and the limit of quantitation was 0.03 pg·μL^-1. The residual Pichia pastoris DNA content in ornithine aspartate bulk of conbercept was far less than 10 ng per dose. CONCLUSION The method can be used for the quantitative determination of Pichia pastoris DNA residues in ornithine aspartate.
出处
《中国现代应用药学》
CAS
CSCD
北大核心
2018年第8期1158-1161,共4页
Chinese Journal of Modern Applied Pharmacy
