Department of Forensic Medicine, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-Ku, Tokyo, 113-8421, Japan.
Department of Forensic Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Saitama, 350-0495, Japan.
J Nat Med. 2019 Jan;73(1):173-178. doi: 10.1007/s11418-018-1261-3. Epub 2018 Oct 29.
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 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 600 bp 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.
我们建立了广泛适用的 DNA 提取方法，以鉴定来源于动物的生药的起源。检查了包括 17 种来源于动物的生药在内的 21 个样本。通过调整 QIAamp DNA Mini 试剂盒，从大多数生药中提取 DNA。通过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。对于高钙含量的生药，如牡蛎壳和乌贼骨，先用 EDTA 脱钙处理，然后应用蛋白酶处理，成功地进行了 DNA 提取。海耳壳不能用 EDTA 脱钙处理提取 DNA，但用 TBONE EX KIT 成功提取。扩增了线粒体 16S 核糖体 RNA(rRNA)基因区域，并在测序后进行了基本局部比对搜索工具 (BLAST) 分析。除了两种海马中的一种驴胶和龙骨外，所有样品均获得了约 600bp 长的 PCR 产物。基于 BLAST 结果的序列分析确定了所有样品的药物起源，匹配率>97%。此外，16 个样品的匹配率>99%。我们的 DNA 提取方法广泛适用于评价许多来源于动物的生药，并被证明非常有助于鉴定这些药物的起源。
