Abstract
With an internal transcribed spacer of 18 S, 5.8 S and 26 S nuclear ribosomal DNA (nrDNA ITS) as DNA marker, we report a colorimetric approach for authentication of Pseudostellaria heterophylla (PH) and its counterfeit species based on the differentiation of the nrDNA ITS sequence. The assay possesses an unlabelled G-quadruplex DNAzyme molecular beacon (MB) probe, employing complementary sequence as biorecognition element and 1:1:1:1 split G-quadruplex halves as reporter. In the absence of target DNA (T-DNA), the probe can shape intermolecular G-quadruplex structures capable of binding hemin to form G-quadruplex-hemin DNAzyme and catalyze the oxidation of ABTS2− to blue-green ABTS•− by H2O2. In the presence of T-DNA, T-DNA can hybridize with the complementary sequence to form a duplex structure, hindering the formation of the G-quadruplex structure and resulting in the loss of the catalytic activity. Consequently, a UV-Vis absorption signal decrease is observed in the ABTS2−-H2O2 system. The “turn-off” assay allows the detection of T-DNA from 1.0 × 10−9 to 3.0 × 10−7 mol·L−1 (R2 = 0.9906), with a low detection limit of 3.1 × 10−10 mol·L−1. The present study provides a sensitive and selective method and may serve as a foundation of utilizing the DNAzyme MB sensor for identifying traditional Chinese medicines.
Highlights
Pseudostellaria heterophylla (PH), a common traditional Chinese medicine, has been utilized to treat night sweats, hyperirritability, asthenia after illnesses, various lung and spleen diseases [1,2,3,4].many counterfeit species, e.g., Lophatherum gracile (LG), Liriope platyphylla (LP), Ophiopogon japonicus (OJ), Stemona sessilifolia (SS) and Stemona japonica (SJ) [5], are misused in the market owing to their lower cost or erroneous identification
Many counterfeit species, e.g., Lophatherum gracile (LG), Liriope platyphylla (LP), Ophiopogon japonicus (OJ), Stemona sessilifolia (SS) and Stemona japonica (SJ) [5], are misused in the market owing to their lower cost or erroneous identification
In the presence of target DNA (T-DNA), the loop of the molecular beacon (MB) sensor is combined and the stem opens to form a duplex DNA structure hindering the formation of the intermolecular G-quadruplex
Summary
Pseudostellaria heterophylla (PH), a common traditional Chinese medicine, has been utilized to treat night sweats, hyperirritability, asthenia after illnesses, various lung and spleen diseases [1,2,3,4].many counterfeit species, e.g., Lophatherum gracile (LG), Liriope platyphylla (LP), Ophiopogon japonicus (OJ), Stemona sessilifolia (SS) and Stemona japonica (SJ) [5], are misused in the market owing to their lower cost or erroneous identification. Analysis of well-characterized marker compounds is the most popular method for identifying the herbal species utilizing HPLC [7,8], HPCE [9], GC-MS [10,11], NIR spectroscopy [4], etc. Many genetic tools have been utilized for the authentication of herbal species, e.g., RAPD [17], RFLP [18], MASA [18], MAS-PCR [19]. The practicality of these methods varies with their sensitivity, reliability and cost of execution [20]. G-quadruplex molecular beacon (MB) DNAzyme, a label-free and amplified detecting assay, fulfills all these requirements
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