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目的:采用常规PCR及实时荧光PCR方法检测肝素钠粗品及原料药中的猪、牛、羊源性成分。方法:利用土壤基因组DNA提取纯化试剂盒从肝素钠粗品及原料药中提取残留核酸,以其为模板,通过常规PCR及实时荧光PCR法进行动物源性成分鉴定。以提取猪肝、牛心、羊肝的DNA为对照品,验证所建立的常规PCR法及实时荧光PCR法的特异性及灵敏度,并用以上两种方法对3批肝素钠粗品及4批肝素钠原料药进行动物源性成分鉴定。结果:采用的两种PCR方法对猪、牛、羊源性成分的鉴定具有特异性,且灵敏度分别达到pg级及10-1pg级。通过土壤基因组DNA提取纯化试剂盒成功从肝素钠粗品及原料药中提取获得适用于PCR反应的模板DNA,显示7批肝素钠样品均为猪源性产品,且未检出牛、羊源性成分污染。结论:建立了一种快速有效地从肝素钠粗品及原料药中提取DNA的方法,并成功通过两种PCR方法对其猪、牛、羊源性成分进行鉴定,为监督药品规范生产,保证用药安全提供了技术支持。
OBJECTIVE: To detect the components of pig, cattle and sheep in crude heparin and APIs by routine PCR and real-time fluorescence PCR. METHODS: Residual nucleic acid was extracted from crude heparin and crude drug by using soil genomic DNA extraction and purification kit. The PCR products were identified by PCR and real-time PCR. To extract the DNA of porcine liver, beef heart and goat liver as reference substance, the specificity and sensitivity of the established routine PCR and real-time fluorescence PCR were validated, and the three batches of crude heparin sodium and four batches of heparin sodium Raw material for animal-derived ingredients identification. Results: The two PCR methods were specific to the identification of porcine, bovine and sheep origin, and their sensitivity reached pg grade and 10-1 pg grade respectively. The template DNA suitable for PCR reaction was successfully obtained from crude heparin and crude drug through soil genomic DNA extraction and purification kit, which showed that all seven batches of heparin sodium samples were porcine-derived products, and no bovine or sheep-derived components were detected Pollution. Conclusion: A rapid and effective method for DNA extraction from crude heparin and crude drug was established. Two methods of PCR were used to identify the components of pig, cattle and sheep. In order to supervise the standardization of drug production and ensure the use of drugs Security provided technical support.