Rapid Authentication of Bupleurum Species using an Array of Immobilized Sequence-Specific Oligonucleotide Probes

Abstract

Standardization and modernization of Chinese medicinal herbs are limited partially by misidentification of processed materials. Our goal was to develop an efficient method for verification of Chinese medicinal herbs, based on the variable sites of the rDNA internal transcribed spacer (ITS) region. We analyzed sequence differences in ITS of three Bupleurum species, B. kaoi Liu Chao et Chuang, B. falcatum L. and B. chinense DC., and developed a rapid detection method using a sequence-specific oligonucleotide probe (SSOP) array. The SSOP array, composed of poly-T tailed sequence-specific oligonucleotides, was hybridized to the digoxigenin (DIG)-labeled target ITS DNA of the Bupleurum species. The detected signals corresponded precisely to the specific sequences. This array provides a reliable and economical method for authenticating a large number of Chinese medicinal herbs. The short duration of the procedure (within 30 h) makes it an especially useful tool in verifying processed plant material.

Abbreviations

CSPD:disodium 3-{4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclodecan]-4-yl}phenyl phosphate

DIG:digoxigenin

GCG:genetics computer group

ITS:inernal transcribed spacer

PCR:polymerase chain reaction

rDNA:ribosomal DNA

RFLP:restriction fragment length polymorphism

SSPO:sequence-specific oligonucleotide probe

TdT:terminal deoxyribonucleotidyl transferase

Tm:melting temperature

Key words

Authentication - Bupleurum - Umbelliferae - ITS - SSOP

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