实时荧光定量PCR法检测门冬氨酸鸟氨酸原料药中毕赤酵母菌DNA残留量

Detection of Residual DNA of Pichia Pastorisin in Ornithine Aspartate by Real-time PCR

关键词:  门冬氨酸鸟氨酸  毕赤酵母菌  DNA残留  实时荧光定量PCR

Key words:  ornithine aspartate  Pichia pastoris  residual DNA  Real-time PCR

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^-1内线性良好（r>0.99），定量限为0.03 pg·μL^-1；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。

Abstract:

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 r>0.99, 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.