Establishment of widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals using molecular techniques
Establishment of widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals using molecular techniques
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DOI：
10.1007/s11418-018-1261-3
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发表时间：
2019-01-01
影响因子：
3.3
通讯作者：
Saito, Kazuyuki
中科院分区：
文献类型：
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作者：
Nakanishi, Hiroaki;Yoneyama, Katsumi;Saito, Kazuyuki
我们建立了广泛适用的DNA提取方法来鉴定动物源性药材的来源。共检测了21个样品，包括17种动物源性药材。通过调整QIAamp（（R））DNA Mini Kit从大多数生药中提取DNA。在应用蛋白酶处理后，使用苯酚去除杂质成功地进行了DNA提取。采用乙二胺四乙酸（EDTA）脱钙处理，对牡蛎壳、乌贼骨等高钙药材进行蛋白酶处理，成功地提取了DNA。用EDTA脱钙法不能从海贝壳中提取DNA，但用TBONE EX试剂盒成功地提取了DNA。扩增线粒体16S核糖体RNA（rRNA）基因区域，测序后进行基本局部比对搜索工具（BLAST）分析。聚合酶链反应（PCR）扩增产物的长度约为600 bp的所有样品中，除了驴胶，两个海马之一，和龙骨。通过基于BLAST结果的序列分析确定所有样品中的药物来源，并且匹配率> 97%。16个样本的匹配率> 99%。我们的DNA提取方法广泛适用于许多动物源性药材的评价，并证明对鉴定此类药物的来源非常有用。
We established widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals. Twenty-one samples including 17 kinds of crude drug derived from animals were examined. DNA was extracted from most of the crude drugs by adjustment of the QIAamp((R)) DNA Mini Kit. DNA extraction was performed successfully using phenol to remove impurities after applying a proteinase treatment. DNA extraction was performed successfully by decalcification treatment using ethylenediaminetetraacetic acid (EDTA), before applying the proteinase treatment for crude drugs having high calcium content, such as those from oyster shell and cuttlefish bone. DNA could not be extracted from sea-ear shell using the EDTA decalcification treatment, but was extracted successfully using a TBONE EX KIT. The mitochondrial 16S ribosomal RNA (rRNA) gene region was amplified, and Basic Local Alignment Search Tool (BLAST) analysis was performed after sequencing. Polymerase chain reaction (PCR) products of approximately 600bp in length were obtained from all samples except donkey glue, one of the two seahorses, and longgu. Drug origins were determined in all samples by sequence analysis based on the BLAST results, and match rates were >97%. Moreover, 16 samples had a match rate >99%. Our DNA extraction methods were widely applicable to evaluation of many crude drugs derived from animals, and proved very useful for identifying the origins of such drugs.