Real-time_PCR法检测注射用门冬酰胺酶制剂中大肠杆菌核苷酸残留

目的：建立可定量检测注射用门冬酰胺酶制剂中大肠杆菌核苷酸残留量的实时荧光定量PCR（Realtime PCR）方法，用于注射用门冬酰胺酶制剂中大肠杆菌核苷酸残留的质量控制。方法：选择大肠杆菌23S核糖体RNA基因为靶基因设计扩增引物，以ABI 7500 FAST平台为基础，建立基于SYBR Green荧光染料的Real-time PCR检测方法。结果：该法检测大肠杆菌核苷酸质量浓度在0.009 35~93 500 ng·mL^-1范围内线性良好，标准曲线的相关系数为0.999，定量限为0.009 35 ng·mL^-1。应用该法对2批注射用门冬酰胺酶制剂进行测定，均未检出大肠杆菌核苷酸残留。结论：该方法可用于注射用门冬酰胺酶制剂中大肠杆菌核苷酸残留的定量测定。

Objective: To establish a Real-time PCR method for quantitative detection of the E. coli nucleotide residues in asparaginase for injection, so as to control the quality of asparaginase for injection.Methods: A Realtime PCR method based on SYBR Green fluorescent dye was established based on the ABI 7500 FAST platform, and the 23S ribosomal RNA gene of E. coli was used as target gene design primer.Results: The calibration curve was linear over the range of 0.009 35 to 93 500 ng·mL^-1, the correlation coefficient was 0.999, and the limit of quantitation was 0.009 35 ng·mL^-1. Using this method to detect two batches of asparaginase for injection, no E. coli nucleotide residues were detected.Conclusion: The method can be used for the quantitative determination of E. coli nucleotide residues in asparaginase for injection.

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