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

Abstract

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.