蒲公英基原真偽之快速鑑定方法－Allele-specific PCR及ISSR標誌

Allele-specific PCR and ISSR Marker Used in Rapid Detection of Adulteration Species of Taraxacum Mongolicum

袁秋英(Chiou-Ing Yuan) ； 林李昌(Li-Chang Lin) ； 林志鍵(Chih-Chien Lin) ； 郭昭麟(Chao-Lin Kuo) ； 蔣慕琰(Mou-Yen Chiang)

《作物、環境與生物資訊》 6卷3期 (2009/09) Pp. 183-191

https://doi.org/10.30061/CEB.200909_6(3).0005

摘要

中草藥之研發與運用漸受矚目，然而藥材基原常出現偽品或代用品，易影響療效，甚至產生中毒問題。近年分子標誌已普遍應用於植物物種之親緣研究，其中比對基原植物基因組核酸之差異，也漸成為鑑定中草藥真偽的有利證據之一。本研究室已完成蒲公英(Taraxacum mongolicum Hand.-Mazz.)及其偽品基原5.8S rRNA-ITS序列，並發表PCR-RFLP檢測技術，此次研究再延伸發展為等位基因特異性之聚合酶鏈鎖反應(allele-specific polymerase chain reaction; AS-PCR)及簡單重複序列間標誌(intersimple sequence repeat; ISSR)之快速檢測方法。經由比對臺灣蒲公英(Taraxacum formosanum Kitanlura)、西洋蒲公英(T. officinale Wiggers)及蒲公英(T. mongolicum)等3種蒲公英以及兔仔菜[Ixeris chinensis (Thunb.) Nakai]、鵝仔草(Pterocypsela indica L.)、苦滇菜(Sonchus oleraceus L.)、黃鵪菜(Youngia japonica L.)、刀傷草[I. laevigata (Bl.) Sch.]等5種偽品基原5.8S rRNA-ITS序列之異同，設計AS-PCR的特異性引子，利用multiplex PCR可於臺灣蒲公英、西洋蒲公英、蒲公英及蒲公英屬基原分別增幅194bp、586bp、402bp及457bp核酸片段，5種偽品基原則否。3種蒲公英及5種偽品基原genomic DNA以ISSR編號UBC-857為引子，經一次PCR增幅，即可分別產生2-6條不同長度核酸的電泳圖譜，具有鑑別蒲公英及偽品基原之功效。本研究所建立之AS-PCR及ISSR標誌檢測方法，可快速、有效及正確區別蒲公英藥材基原之真偽，作為中草藥品質控管及基原鑑定之依據。

關鍵字

蒲公英 ； 中藥 ； 分子標誌 ； 核糖體核酸間隔區 ； 等位基因特異性之聚合酶鏈鎖反應 ； 簡單重複序列間標誌。

並列摘要

Substitutes and adulterants of traditional Chinese medicine (TCM) materials are often introduced intentionally or accidentally. It may seriously affect the regular therapeutic effects, even leading to life-threatening poisoning. DNA markers have been widely used for identification of plant species in recent decade. In this study, a previously published polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for molecular authentication of Taraxacum mongolicum was modified to a rapid allelic specific PCR (AS-PCR) and inter-simple sequence repeat (ISSR) markers. The specific primers of AS-PCR were designed from the 5.8S rRNA-ITS nucleotide polymorphism to differentiate three Taraxacum species and five adulterant species, via multiplex PCR to produce unique 194bp, 586bp, 402bp and 457bp single bands for T. formosanum, T. officinale, T. mongolicum and Taraxacum spp., respectively. Among the selected ISSR primers, only UBC857 can significantly distinguish three Taraxacum species and five adulterant species by 2-6 different bands profiles. Those two novel AS-PCR and ISSR markers provide the effective and accurate identification of Taraxacum species.

並列關鍵字

Taraxacum mongolicum ； Traditional Chinese medicine TCM ； Molecular marker ； Internal transcribed spacer ITS ； Allele-specific polymerase chain reaction AS-PCR ； Intersimple sequence repeat ISSR