肝素样品前处理方法比较及荧光定量PCR法鉴定肝素原料种属来源

Comparison of crude heparin pretreatment methods and identification of crude heparin from various species origins by quantitative PCR

目的比较肝素原料的3种前处理方法,即磁珠吸附法、琼脂糖凝胶回收法和肝素酶降解法的优劣,并对肝素原料的种属来源进行鉴别。方法分别用磁珠提取试剂盒、琼脂糖凝胶回收试剂盒和肝素酶对肝素原料进行前处理,用商业化的猪、牛、羊种属鉴别试剂盒进行检测。采用荧光定量PCR法对3批肝素原料进行种属来源的鉴别,同时对猪源肝素中掺入的不同比例羊源肝素进行检出限的检测。结果 3种不同的前处理方法以肝素酶降解法最快捷、最省时和最经济;种属来源鉴别结果准确。荧光定量PCR法检测猪源肝素中掺入羊源肝素的检出限是0.01%。结论肝素酶降解法能简捷、快速、经济的解决肝素原料中样品前处理的技术难点,结合商业化的猪、牛、羊鉴别试剂盒与荧光定量PCR仪能够简便、快速、准确的对肝素原料种属来源进行鉴别。

Objective To compare magnetic beads kit,agrose gel recovery kit and heparinase I three methods to purify the micro DNA from crude heparin,then use q-PCR to identify the species origins and select the best method. Methods Using magnetic beads kit,agrose gel recovery kit and heparinase I to purify micro DNA from crude heparin and combined the porcine,bovine and ovine identification kits to identify the species origins and conformed the minimum detection limit of different percentage of ovine crude heparin in porcine crude heparin. Results Three pretreatment methods all can solve the pretreatment difficulties and we found that the haparinase was the best method; the minimum detection limit was 0. 01% of ovine crude heparin in porcine crude heparin. Conclusion The heparinase method is the best pretreatment method and can successfully solve the pretreatment difficulties. Heparinase combine the porcine,bovine and ovine identification kits can identify the species origins from crude heparin.