利用Nested PCR-DNA定序方法鑑定肉蓯蓉藥材基原

Identification of Cistanches Herba Based on psbA-trnH Intergenic Spacer Region Sequence by Nested PCR-DNA Sequencing Method

依據臺灣中藥典規範肉蓯蓉藥材應使用列當科植物肉蓯蓉Cistanche deserticola Y. C. Ma或管花肉蓯蓉Cistanche tubulosa（Schrenk）Wight之帶鱗葉的乾燥肉質莖。由於各地區用藥習慣不同及外型相似等原因常出現同名異物或同物異名的情況，為避免藥材誤用影響到用藥安全與有效性，本研究從DNA層面確認肉蓯蓉藥材之基原正確性。收集來自各地共計40件藥材與14件製劑檢體，利用肉蓯蓉藥材正品基原之葉綠體psbA-trnH基因間隔區（chloroplast psbA-trnH intergenic spacer）的序列差異設計特異性引子對，以Nested PCR（Nested polymerase chain reaction）反應續以DNA定序方法進行基原鑑別。肉蓯蓉Nested PCR反應產物經膠片電泳分析可得約252鹼基對（base pair）之產物，管花肉蓯蓉Nested PCR反應產物可得約386鹼基對之產物，利用電泳擴增片段大小可初步區分肉蓯蓉與管花肉蓯蓉，續以DNA定序確認物種。檢體測試結果證明本方法能專一地鑑定出藥材與製劑是否使用正確基原的肉蓯蓉藥材，期能應用在中藥檢驗及品質管控上，以保障用藥安全及國民健康。 Cistanches Herba is the dried succulent stem of Cistanche deserticola Y. C. Ma or Cistanche tubulosa (Schrenk) Wight in the family Orobanchaceae. The morphological similarities of other Cistanche species to Cistanches Herba has caused high chance of misidentification and misuse. For the purpose of detecting and avoiding material misusing, this study aimed on the correctness of Cistanches Herba form DNA level. Specific primers based on the differences of psbA-trnH (chloroplast psbA-trnH intergenic spacer) sequence were designed. Nested polymerase chain reaction (Nested PCR) and DNA sequencing methods were developed. The developed methods were used for the Cistanches Herba identification. There were 40 raw herbs and 14 traditional Chinese medicine (TCM) preparations surveyed and analyzed in the study. The results showed that the gel electrophoresis for DNA fragment sizes of Nested PCR products was able to differentiate two herbal origins accurately. The Nested PCR amplified fragments for C. deserticola and C. tubulosa were 252 bps and 386 bps, respectively. Nested PCR-DNA sequencing method offered high selectivity and specificity for the rapid identification of Cistanches Herba. This study aimed to facilitate TCM quality control and protect public health.