Differentiating Authentic Adenophorae Radix from Its Adulterants in Commercially-Processed Samples Using Multiplexed ITS Sequence-Based SCAR Markers

Abstract

Determining the precise botanical origin of a traditional herbal medicine is important for basic quality control. In both the Chinese and Korean herbal pharmacopoeia, authentic Adenophorae Radix is defined as the roots of Adenophora stricta and Adenophora triphylla. However, the roots of Codonopsis lanceolata, Codonopsis pilosula, and Glehnia littoralis are frequently distributed as Adenophorae Radix in Korean herbal markets. Unfortunately, correctly identifying dried roots is difficult using conventional methods because the roots of those species are morphologically similar. Therefore, we developed DNA-based markers for the identification of authentic Adenophorae Radix and its common adulterants in commercially-processed samples. To develop a reliable method to discriminate between Adenophorae Radix and its adulterants, we sequenced the nuclear ribosomal DNA internal transcribed spacers (nrDNA-ITS) and designed sequence-characterized amplified region (SCAR) primers specific to the authentic and adulterant species. Using these primers, we developed SCAR markers for each species and established a multiplex-PCR method that can authenticate the four herbal medicines in a single PCR reaction. Furthermore, we confirmed that commercially-processed herbal medicines, which often have degraded DNA, could be assessed with our method. Therefore, our method is a reliable genetic tool to protect against adulteration and to standardize the quality of Adenophorae Radix.

Development of conventional PCR and real‐time PCR assays to discriminate the origins of Chinese pepper oil and herbal materials from Zanthoxylum

“…In contrast to plant DNA barcode regions located in the chloroplast genome, the ITS (or ITS2) locus is common to all eukaryotic genomes, and is present in the nucleus as tandem repeats . Although botanical taxonomists recommend analysis of DNA barcodes for accurate species identification, such assays are cumbersome and time‐consuming, involving DNA amplification, electrophoresis, gel rescue and sequence analysis . In recent years, sequence characterized amplified region (SCAR) markers based on species‐specific sequences obtained from DNA barcodes have been used to discriminate species in raw food materials and herbal medicines …”

“…12 Although botanical taxonomists recommend analysis of DNA barcodes for accurate species identification, such assays are cumbersome and time-consuming, involving DNA amplification, electrophoresis, gel rescue and sequence analysis. [13][14][15] In recent years, sequence characterized amplified region (SCAR) markers based on species-specific sequences obtained from DNA barcodes have been used to discriminate species in raw food materials and herbal medicines. 16,17 Raw food materials are altered by various manufacturing techniques involving physical, chemical or biochemical treatments.…”

A Brief Review of Molecular Markers to Analyse Medically Important Plants

“…Investigation of new DNA barcode regions using plastid genome sequencing [49,50,51]. In the first method, RAPD technique, either alone or in combination with other techniques is extensively used for the genetic analyses of different medicinal plants, and even other organisms [52,53]. SCAR markers derived from the molecular cloning of RAPD fragments in medicinal plants are commonly applied because of its stability, sensitivity, and reliability [54].…”

Rapid and Simple Species Identification of Cicada Exuviae Using COI-Based SCAR Assay

“…Sequence-characterized amplified-region (SCAR) marker assays are PCR-based assays using 15-30 bp primers with nucleotide-sequence specificity; they enable rapid, simple, cheap, reliable, and reproducible species identification [28,29]. SCAR marker assays can be used to distinguish closely related samples by amplifying only target samples using specific primers under high annealing temperatures [28,[30][31][32]. Samples can be differentiated on the basis of amplification results and length polymorphisms [28,30,31].…”

“…SCAR marker assays can be used to distinguish closely related samples by amplifying only target samples using specific primers under high annealing temperatures [28,[30][31][32]. Samples can be differentiated on the basis of amplification results and length polymorphisms [28,30,31]. In animal taxa, mitochondrial DNA is commonly used for species identification, and the cytochrome c oxidase subunit I (COI) gene serves as a primary barcode region [33,34].…”