Visual Detection of Cucumber Green Mottle Mosaic Virus Based on Terminal Deoxynucleotidyl Transferase Coupled with DNAzymes Amplification

Abstract

A simple, rapid, and sensitive visual detection method for observing cucumber green mottle mosaic virus was reported based on the template-independent polymerization activity of terminal deoxynucleotidyl transferase (TdT), coupled with the cascade amplification of Mg2+-dependent DNAzyme and hemin/G-quadruplex DNAzyme. Briefly, the hybridized dsDNA of T1/P1 was cut into two parts at its position of 5′-AA↓CG↑TT-3′ by the restricted enzyme AcII. The longer, newborn fragment originating from P1 was tailed at its 3’-end by oligo dG, and an intact enzymatic sequence of Mg2+-dependent DNAzyme was generated. The substrate sequence in the loop segment of the hairpin probe (HP) hybridized with the newborn enzymatic sequence and was cleaved into two parts in the presence of Mg2+. The locked G-quadruplex sequence in the stem segment of the HP was released, which catalyzed the oxidation of ABTS2- in the presence of H2O2, and the resulting solution turned green. A correlation between the absorbance and concentration of T1 was obtained in a range from 0.1 pM to 2 nM, with a detection limit of 0.1 pM. In addition to promoting a lower detection limit and shorter monitoring time, this method also demonstrated an excellent selectivity to single or double nucleotide changes. Therefore, the designed strategy provided a rapid and efficient platform for viral inspection and plant protection.

Visual Detection of SARS-CoV-2 RNA by Conventional PCR-Induced Generation of DNAzyme Sensor

“…Previous reports have employed a similar strategy to detect both human and plant viruses (Yang et al, 2017;Kim et al, 2018;Wang et al, 2019), and a recent report has suggested the potential use of DNAzyme sensors for SARS-CoV-2 detection (Xi et al, 2020). We here demonstrate the utility of this approach for SARS-CoV-2.…”

Synthesis of enzymatic hydrogels and their application in biosensors

“…图 17 基于OTA适配体和TDT介导超长聚胸腺嘧啶转换的 Cu NPs的OTA荧光测定示意图 [49] (网络版彩图) 图 19 基于近端双向酶修复扩增的ATP荧光检测原理图 [51] (网络版彩图) 图 18 末端脱氧核苷酸转移酶(TdT)辅助扩增策略在黄瓜 绿斑驳花叶病毒视觉检测中的应用原理图 [50] (网络版彩图) [46] (网 络版彩图) 图 21 基于NIEP的E-DNA生物传感原理 [52] (网络版彩图)…”

“…Figure18 Schematic diagram of the application of terminal deoxynucleotidyl transferase (TdT) assisted amplification strategy in the visual detection of cucumber green mottle mosaic virus[50] (color online).…”

Visual detection of Fusarium proliferatum based on dual-cycle signal amplification and T5 exonuclease

“…31 They are used in a variety of label-free biosensors due to the advantages of simple use, cost-effectiveness, easy modication and acquisition. 32,33 G-quadruplex-related strategies are also coupled with signal amplication steps, for example strand displacement amplication 34,35 and RCA 36 to elevate the sensitivity. RCA produces large weight ssDNA with G-rich sequences using circular ssDNA as template bearing trans-complementary G-quadruplex sequences.…”