Dataset Information

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 ABTS^2- in the presence of H?O?, 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.

SUBMITTER: Wang Y

PROVIDER: S-EPMC6471243 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Publications

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

Wang Ying Y Liu Jing J Zhou Hong H

Sensors (Basel, Switzerland) 20190314 6

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 origin ...[more]

PMID: 30875853

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Project description:BackgroundSince it was first reported in 1935, Cucumber green mottle mosaic virus (CGMMV) has become a serious pathogen in a range of cucurbit crops. The virus is generally transmitted by propagation materials, and to date no effective chemical or cultural methods of control have been developed to combat its spread. The current study presents a preliminary analysis of the pathogenic mechanisms from the perspective of protein expression levels in an infected cucumber host, with the objective of elucidating the infection process and potential strategies to reduce both the economic and yield losses associated with CGMMV.MethodsIsobaric tags for relative and absolute quantitation (iTRAQ) technology coupled with liquid chromatography-tandem mass spectrometric (LC-MS/MS) were used to identify the differentially expressed proteins in cucumber plants infected with CGMMV compared with mock-inoculated plants. The functions of the proteins were deduced by functional annotation and their involvement in metabolic processes explored by KEGG pathway analysis to identify their interactions during CGMMV infection, while their in vivo expression was further verified by qPCR.ResultsInfection by CGMMV altered both the expression level and absolute quantity of 38 proteins (fold change >0.6) in cucumber hosts. Of these, 23 were found to be up-regulated, while 15 were down-regulated. Gene ontology (GO) analysis revealed that 22 of the proteins had a combined function and were associated with molecular function (MF), biological process (BP) and cellular component (CC). Several other proteins had a dual function with 1, 7, and 2 proteins being associated with BP/CC, BP/MF, CC/MF, respectively. The remaining 3 proteins were only involved in MF. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 18 proteins that were involved in 13 separate metabolic pathways. These pathways were subsequently merged to generate three network diagrams illustrating the interactions between the different pathways, while qPCR was used to track the changes in expression levels of the proteins identified at 3 time points during CGMMV infection. Taken together these results greatly expand our understanding of the relationships between CGMMV and cucumber hosts.ConclusionsThe results of the study indicate that CGMMV infection significantly changes the physiology of cucumbers, affecting the expression levels of individual proteins as well as entire metabolic pathways. The bioinformatic analysis also identified several pathogenesis-related (PR) proteins that could be useful in the development of disease-resistant plants.

Dataset Information

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

Publications

Visual Detection of <i>Cucumber Green Mottle Mosaic Virus</i> Based on Terminal Deoxynucleotidyl Transferase Coupled with DNAzymes Amplification.

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