The cauliflower mosaicvirus 35S (CaMV35S), as the most common promoter, has a usage rate of 70 % in transgenic crops, especially in dicots. Herein, a visual detection method of rolling circle amplification (RCA) combined with DNAzyme technology was developed for the determination of CaMV35S gene. A template sequence of the G-quadruple gene was designed for combining with the target gene CaMV35S. When the target gene existed, the RCA reaction occurred through the action of the enzyme, and the product combined with hemin to generate DNAzyme, which catalyzed the color reaction of 2′-hydrazine-bis-3-ethylbenzothiazolin-6-sulfonic acid (ABTS). Under the optimal experimental conditions, the linear range is 1 × 10 −13 ∼1 × 10 -8 mol/L, and the detection limit is 3 × 10 -14 mol/L (S/N = 3). The method has been successfully applied to standard samples and practical samples (soybean and tofu). For verifying the feasibility and reliability, real-time fluorescent quantitative PCR method was used to detect the target gene CaMV35S, and the results were in good agreement with those of our method, indicating that our developed method can be used for the determination of actual samples.
Yaqi Li,Li Sun,Qian Liu,En Han,Nan Hao,Liuping Zhang,Shanshan Wang,Jianrong Cai,Kun Wang
A methodology for detection of the Cauliflower Mosaic Virus 35S(CaMV35S) promoter was developed to distinguish transgenic from non-transgenic soybean samples by using photoelectrochemical (PEC) biosensor. In this PEC biosensing system, the as-prepared gold nanoparticles-reduced graphene oxide acted as a nanocarrier to immobilize the thiol-functional probe (probe1), and the SiO 2 @CdTe quantum dots (QDs) core-shell nanoparticles tagged with the amino-functional probe (probe2) acted as signal indicators, respectively. In the presence of target DNA (tDNA) of CaMV35S, the binding of tDNA with probe1 and probe2 through the high specific DNA hybridization led to the fabrication of sandwich structure, and thus the high loading of the signal indicators SiO 2 @CdTe QDs at the electrode surface, which increased the PEC signal. The increased PEC signal depended on the concentration of tDNA, and a wide linear range from 0.1 pM to 0.5 nM with low detection limit of 0.05 pM was obtained. In addition, the PEC biosensor has been successfully used for discriminating transgenic soybean from non-transgenic samples, which was consistent with the polymerase chain reaction (PCR) results, suggesting the proposed PEC biosensor is a feasible tool for the further daily genetically modified organism detection.
In this work, a dumbbell-shaped bi-acetylferrocene ethylenediamine complex, (AFc) 2 -en (AFc = acetylferrocene, en = ethylenediamine) was synthesized and its interaction with double-stranded DNA (dsDNA) was comprehensively studied by electrochemical methods on a gold electrode. The experimental results reveal that the highly electroactive (AFc) 2 -en complex can interact with dsDNA via a specific bis-groove binding mode. The binding constant and binding site size between the two species were determined to be 3.7 × 10 5 L mol −1 and 4.2, respectively. The surface-based studies further show that (AFc) 2 -en presents the stronger electrochemical response on dsDNA modified electrode than on the single-stranded DNA (ssDNA) modified electrode, suggesting that the complex can well distinguish dsDNA from ssDNA. Based on this characteristic, the bi-acetylferrocene complex was utilized as an electrochemical hybridization indicator in a DNA biosensor for the detection of oligonucleotides from CaMV35S promoter gene. The specificity experiments indicate that the biosensor can well discriminate the complementary sequence from the base-mismatched and the non-complementary sequences. The complementary target sequence can be quantified over a wide range from 1.0 × 10 −14 mol L −1 to 1.0 × 10 −6 mol L −1 with a detection limit of 2.0 × 10 −15 mol L −1 .
危险品化学品经营许可证（带存储）