{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T17:57:22Z","timestamp":1790704642813,"version":"4.1.0"},"reference-count":24,"publisher":"IMR Press","issue":"9","license":[{"start":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T00:00:00Z","timestamp":1789689600000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Gansu Provincial Pharmaceutical Regulatory Science Research Project","award":["2023GSMPA011"],"award-info":[{"award-number":["2023GSMPA011"]}]},{"name":"Gansu Provincial University Teacher Innovation Fund Project","award":["2023A-279"],"award-info":[{"award-number":["2023A-279"]}]},{"name":"Gansu Provincial University Teacher Innovation Fund Project","award":["2024A-292"],"award-info":[{"award-number":["2024A-292"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmazie"],"abstract":"<jats:p>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\u2032 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\u2032-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 \u03bcmol\/L, an annealing temperature of 60 \u2103, and 35 amplification cycles; for the colorimetric reaction, final concentrations of 5 \u03bcmol\/L hemin and 2 mmol\/L ABTS, with denaturation at 95 \u2103 for 8 min, and incubation at 4 \u2103 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\/\u03bcL, with a reliable naked-eye visual threshold at \u22652 ng\/\u03bcL and a validated detection range extending to at least 50 ng\/\u03bcL 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.<\/jats:p>","DOI":"10.31083\/pharmazie53662","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T17:05:53Z","timestamp":1790701553000},"source":"Crossref","is-referenced-by-count":0,"title":["Visual Differentiation of Bupleurum marginatum var. stenophyllum From Bupleurum chinense DC. Using a PCR-Coupled G-Quadruplex Colorimetric Assay"],"prefix":"10.31083","volume":"81","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-9030-1535","authenticated-orcid":true,"given":"Shunjie","family":"Yuan","sequence":"first","affiliation":[{"name":"Department of Pharmacology, Lanzhou Institute for Food and Drug Control, 730050 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fuxiang","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Lanzhou Institute for Food and Drug Control, 730050 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenhui","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Lanzhou Institute for Food and Drug Control, 730050 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Li","sequence":"additional","affiliation":[{"name":"Gansu Food Inspection and Research Institute (Gansu Animal-derived Food Gene Testing Center), 730300 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhen","family":"Tian","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Lanzhou Institute for Food and Drug Control, 730050 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenjing","family":"Bai","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Lanzhou Institute for Food and Drug Control, 730050 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bin","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Lanzhou Institute for Food and Drug Control, 730050 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaojun","family":"Song","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Lanzhou Institute for Food and Drug Control, 730050 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoxia","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Traditional Chinese Medicine, Lanzhou Institute for Food and Drug Control, 730050 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liyan","family":"Fu","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Lanzhou Institute for Food and Drug Control, 730050 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-6032-5381","authenticated-orcid":true,"given":"Jinli","family":"Yue","sequence":"additional","affiliation":[{"name":"Gansu Vocational College of Agriculture, School of Health Care and Education, 730030 Lanzhou, Gansu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"16242","published-online":{"date-parts":[[2026,9,18]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","unstructured":"Tian Y, Guo J, Jiang X, Lu H, Xie J, Zhang F, et al. 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