Development of the species-specific multiplex PCR and DNA sequencing methods for rapid authentication of Isatidis Folium and its adulterants

Yung-Chuan Hsieh ^a, Ming-Sian Wu ^a, Hui-Chun Lee ^a, Chia-Yun Hsieh ^a, Shih-Shan Huang ^a, Chia-Fen Tsai ^b, Ya-Tze Lin ^a,*, Mei-Chih Lin ^a, Su-Hsiang Tseng ^a, Der-Yuan Wang ^a

a Division of Research and Analysis, Food and Drug Administration, Ministry of Health and Welfare, No.161-2 Kunyang St., Nangang Dist., Taipei 11561, Taiwan

b Southern Center for Regional Administration, Food and Drug Administration, Ministry of Health and Welfare, No.180, Zihyou 2nd Rd., Zuoying Dist., Kaohsiung 81358, Taiwan

Isatis indigotica Fort. (family Cruciferae), is an herb widely used in traditional herbal medicine and its dried leave was named “ISATIDIS FOLIUM”. Baphicacanthus cusia (Ness) Bremek. and Polygonum tinctorium Ait. are commonly misused as ISATIDIS FOLIUM in Chinese Medicine pharmacy. For the purpose of being not misused, specific primers based on the sequence difference of chloroplast trnH-psbA intergenic spacer were designed and multiplex polymerase chain reaction method (multiplex PCR) was developed. In this study, 29 original herbal materials were analyzed and our results show that DNA size after multiplex PCR was able to distinguish variations between three herbs. DNA fragments of 464, 297, 170 base pairs (bps) were represented for I. indigotica and B. cusia and P. tinctorium, respectively. In conclusion, our investigations demonstrate that molecular identification method provides more accurate results for medicinal plants detection and good quality control of ISATIDIS FOLIUM.

Keywords: DNA sequencing, ISATIDIS FOLIUM, Multiplex PCR, TrnH-psbA intergenic spacer

https://doi.org/10.38212/2224-6614.3354

(https://www.jfda-online.com/journal/vol29/iss2/9)