A quantitative PCR method to quantify ruminant DNA in porcine crude heparin.
A quantitative PCR method to quantify ruminant DNA in porcine crude heparin.
复制标题
DOI：
10.1007/s00216-010-4362-8
复制
发表时间：
2011-01
影响因子：
4.3
通讯作者：
Workman, Wesley E.
中科院分区：
文献类型：
--
作者：
Concannon, Sean P.;Wimberley, P. Brett;Workman, Wesley E.
关键词：
肝素是从猪肠中提取的一种著名的糖胺聚糖。提高对动物源性药物中传染性海绵状脑病的警惕，需要采取措施防止反刍动物将肝素引入供应链。定量聚合酶链反应（PCR）的敏感性、特异性和精确性使其成为筛选牛、羊和羊嘌呤来源的肝素原料的优越分析平台。与反刍动物Bov-A2短穿插核元件（SINE）位点同源的定量PCR探针和引物（Mendoza-Romero等）。J. Food Prot. 67:550-554, 2004)在定量PCR方法中显示出与牛、羊和山羊DNA目标几乎相同的亲和力，而与猪DNA没有交叉反应性。还开发了第二种PCR引物和探针，专门用于猪PRE1 SINE序列，以量化猪的背景DNA水平。脱氧核糖核酸的提取和纯化是没有必要的原料肝素样品的分析，虽然消化样品的肝素酶采用。该方法在反刍动物肝素DNA的定量范围为0.3 ~ 3000 ppm。方法的验证参数包括准确度、重复性、精密度、特异性、范围、定量限和线性。
Heparin is a well-known glycosaminoglycan extracted from porcine intestines. Increased vigilance for transmissible spongiform encephalopathy in animal-derived pharmaceuticals requires methods to prevent the introduction of heparin from ruminants into the supply chain. The sensitivity, specificity, and precision of the quantitative polymerase chain reaction (PCR) make it a superior analytical platform for screening heparin raw material for bovine-, ovine-, and caprine-derived material. A quantitative PCR probe and primer set homologous to the ruminant Bov-A2 short interspersed nuclear element (SINE) locus (Mendoza-Romero et al. J. Food Prot. 67:550–554, 2004) demonstrated nearly equivalent affinities for bovine, ovine, and caprine DNA targets, while exhibiting no cross-reactivity with porcine DNA in the quantitative PCR method. A second PCR primer and probe set, specific for the porcine PRE1 SINE sequence, was also developed to quantify the background porcine DNA level. DNA extraction and purification was not necessary for analysis of the raw heparin samples, although digestion of the sample with heparinase was employed. The method exhibits a quantitation range of 0.3–3,000 ppm ruminant DNA in heparin. Validation parameters of the method included accuracy, repeatability, precision, specificity, range, quantitation limit, and linearity.
登录
查看更多内容
影响因子：
2.9
作者：
Walker, JA;Hughes, DA;Batzer, MA
通讯作者：
Batzer, MA
影响因子：
2.9
作者：
Houiste, Celine;Auguste, Cecile;Anger, Pascal
通讯作者：
Anger, Pascal
影响因子：
3.8
作者：
Will, RG
通讯作者：
Will, RG
影响因子：
3.3
作者：
Huang, Qing;Yao, Chun-Yan;Fu, Wei-Ling
通讯作者：
Fu, Wei-Ling
影响因子：
--
作者：
Levieux, A;Rivera, V;Levieux, D
通讯作者：
Levieux, D