Species-specific identification by inhibitor-controlled PCR of ruminant components contaminating industrial crude porcine heparin
Species-specific identification by inhibitor-controlled PCR of ruminant components contaminating industrial crude porcine heparin
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DOI：
10.1016/j.mcp.2006.01.005
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发表时间：
2006-06-01
影响因子：
3.3
通讯作者：
Fu, Wei-Ling
中科院分区：
文献类型：
--
作者：
Huang, Qing;Yao, Chun-Yan;Fu, Wei-Ling
随着牛海绵状脑病的出现，目前药用肝素的来源仅限于猪肠粘膜。为控制工业粗肝素的种属来源，应发展以种属特异性蛋白质或DNA分析为基础的分子生物学方法来鉴定工业粗肝素（ICPH）中可能是污染物的反刍动物成分。由于ICPH中含有的肝素是一种强PCR抑制物质，因此有必要探索ICPH特异性DNA提取方法，并开发可监测PCR抑制物质存在的分析方法。本研究采用7种方法从ICPH中提取DNA，并采用对照引导PCR（IC-PCR）比较了它们去除PCR抑制物质的能力。结果表明，在优化内对照终浓度的基础上，IC-PCR是一种快速、灵敏、高效的检测ICPH中PCR抑制物质的方法，只有琼脂糖凝胶纯化法才能完全去除ICPH中的PCR抑制物质。(c)2006爱思唯尔有限公司保留所有权利。
With the emergence of bovine spongiform encephalopathy, the source of pharmaceutical heparin is currently restricted to porcine intestinal mucosa. To control the species origin of industrial crude heparin, molecular biology methods relying on species-specific protein or DNA analysis should be developed to identify the ruminant components that might be contaminants in industrial crude porcine heparin (ICPH). Because heparin contained in ICPH is a strong PCR inhibitory substance, it is necessary to explore DNA extraction methods specific for ICPH and develop analysis methods that could monitor the presence of PCR inhibitory substances. In the present studies, DNA was extracted from ICPH by seven methods, and their abilities to remove the PCR inhibitory substances were compared using inhibitory-control led PCR (IC-PCR). The results showed that, based on the optimization of the final concentration of the internal processing control (IPC), IC-PCR was a rapid, sensitive and efficient way to monitor the presence of PCR inhibitory substances contained in DNA extracted from ICPH, and only the agarose gel purification method could be used to completely eliminate the PCR inhibitory substances contained in ICPH. (c) 2006 Elsevier Ltd. All rights reserved.