Visual Differentiation of Bupleurum marginatum var. stenophyllum From Bupleurum chinense DC. Using a PCR-Coupled G-Quadruplex Colorimetric Assay.

BACKGROUND

Adulteration of official Bupleurum chinense DC. with the morphologically similar Bupleurum marginatum Wall. ex DC. var. stenophyllum (Wolff) Shan et Y. Li poses safety risks. This study aimed to develop a rapid and visual method for differentiating these two species using a polymerase chain reaction (PCR)-coupled G-quadruplex/DNA enzyme (DNAzyme) colorimetric assay.

METHODS

Using species-specific single nucleotide polymorphism (SNP) loci in the internal transcribed spacer 2 (ITS2) region of ribosomal DNA, we designed forward and reverse primers tagged with the reverse complement of a G-quadruplex-forming sequence at their 5' termini. Following PCR, the target sequences were specifically amplified, generating amplicons flanked by G-quadruplex-forming motifs at both termini. The amplicons were then subjected to thermal denaturation, after which the G-rich sequences folded into G-quadruplex structures in the presence of K+. These structures were subsequently bound to hemin to form a G-quadruplex/hemin DNAzyme with peroxidase-like activity. The DNAzyme catalyzes H2O2-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) to a blue-green product. The PCR amplification and colorimetric reaction conditions were optimized by single-factor gradient experiments.

RESULTS

The optimal conditions were determined as follows: for PCR, a final primer concentration of 0.32 μmol/L, an annealing temperature of 60 ℃, and 35 amplification cycles; for the colorimetric reaction, final concentrations of 5 μmol/L hemin and 2 mmol/L ABTS, with denaturation at 95 ℃ for 8 min, and incubation at 4 ℃ for 20 min. Validation of this assay using 21 Bupleurum samples confirmed that it specifically identified B. marginatum var. stenophyllum, while showing no cross-reactivity with B. chinense, Bupleurum scorzonerifolium Willd., or other congeneric Bupleurum species included in the study. The statistical LOD of the assay was determined to be 1 ng/μL, with a reliable naked-eye visual threshold at ≥2 ng/μL and a validated detection range extending to at least 50 ng/μL with no observable hook effect.

CONCLUSIONS

This assay enables naked-eye readout with high specificity and sensitivity. It provides a promising technical platform for the rapid differentiation of B. marginatum var. stenophyllum from B. chinense, as well as a broadly adaptable research framework for the visual differentiation of congeneric adulterants in traditional Chinese medicinal materials.