荧光定量PCR鉴定肝素粗品种属来源质控方法研究

发布时间：2018-06-27 07:11

本文选题：肝素粗品 + 种属　；参考：《药物生物技术》2016年06期

【摘要】：研究了基于荧光定量PCR方法的肝素粗品种属来源质控方法,用于肝素药品生产企业的原料验收质量控制。该方法采用实时定量PCR法,针对猪、牛、绵羊、山羊基因组特有的基因序列设计引物与探针,采用聚合酶链式反应对目标基因进行扩增,利用扩增曲线的Ct值绘制标准曲线,通过未知样品的Ct值对未知样品初始模板中的基因进行定量。测定时通过肝素酶处理肝素原料,以消除肝素介导的PCR抑制效应。结果:该方法在本实验室条件下具有较好的专属性、准确度、精密度和耐用性:猪基因在30~0.06 ng/μL范围内呈良好线性,牛、绵羊、山羊基因在0.6~0.000 3 ng/μL范围内呈良好线性;猪基因的定量限为0.001 ng/μL,牛、绵羊、山羊基因的定量限为0.000 3 ng/μL。本质控方法较基因组纯化试剂盒提纯方法具有保证被检基因的真实性、原始性、完整性等优点。
[Abstract]:The quality control method of crude heparin species based on fluorescent quantitative PCR (FQ-PCR) was studied, which was applied to the quality control of raw material acceptance in heparin drug manufacturers. In this method, real-time quantitative PCR was used to design primers and probes for gene sequences specific to the genomes of pigs, cattle, sheep and goats. Polymerase chain reaction (PCR) was used to amplify the target gene, and the standard curve was drawn by Ct value of the amplification curve. The genes in the initial template of unknown samples were quantified by the Ct values of unknown samples. Heparinase was used to treat heparin in order to eliminate the effect of heparin-mediated PCR. Results: this method has good specificity, accuracy, precision and durability in our laboratory. The linearity of pig gene is good in the range of 30 ~ 0.06 ng/ 渭 L, and that of cattle, sheep and goat gene is good in the range of 0.6 ~ 0.000 3 ng/ 渭 L; The quantitative limit of porcine gene was 0.001 ng/ 渭 L, and that of cattle, sheep and goat gene was 0.000 3 ng/ 渭 L. The method of essence control has the advantages of ensuring the authenticity, originality and integrality of the tested gene compared with the method of genomic purification kit.
【作者单位】：常州千红生化制药股份有限公司;
【分类号】：R440

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