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Establishment of widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals using molecular techniques

Nakanishi, Hiroaki, Yoneyama, Katsumi, Hayashizaki, Yoshie, Hara, Masaaki, Takada, Aya, Saito, Kazuyuki
Natural medicines 2019 v.73 no.1 pp. 173-178
DNA, EDTA (chelating agent), Sepiidae, Syngnathidae, asses, calcium, drugs, genes, mitochondria, oyster shells, phenol, polymerase chain reaction, proteinases, ribosomal RNA, sequence analysis
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.