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.
Highlights
Cucumber green mottle mosaic virus (CGMMV) belongs to the genus Tobamovirus and has a single-stranded, positive-sense RNA genome of about 6.4 kb [1,2].The infected cucurbit crops exhibit green mottle, mosaic, and uneven symptoms around growing points
CGMMV presents in the leaves, seeds, vines, and roots of infected melon crops and can survive for 10 months in rotting organic matter in the soil
The principle of this assay is illustrated in Scheme 1 and involves two steps: (1) hybridization, digestion, and tailing reaction, and (2) activation of the G-quadruplex structure and colorimetric detection
Summary
Cucumber green mottle mosaic virus (CGMMV) belongs to the genus Tobamovirus (family Cucurbitaceae) and has a single-stranded, positive-sense RNA genome of about 6.4 kb [1,2].The infected cucurbit crops exhibit green mottle, mosaic, and uneven symptoms around growing points. Cucumber green mottle mosaic virus (CGMMV) belongs to the genus Tobamovirus (family Cucurbitaceae) and has a single-stranded, positive-sense RNA genome of about 6.4 kb [1,2]. CGMMV is an economically crucial virus which can be transmitted mechanically or through seeds, resulting in a serious reduction in cucumber, melon, watermelon, and similar crops all over the world. CGMMV presents in the leaves, seeds, vines, and roots of infected melon crops and can survive for 10 months in rotting organic matter in the soil. This indicates that it is very difficult to be eliminated from polluted soil. Strict entry detection is one of the effective measures that can be taken to avoid introducing seeds or seedlings of melon crops bearing CGMMV into the soil
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