Colorimetric detection of genetically modified organisms based on exonuclease III-assisted target recycling and hemin/G-quadruplex DNAzyme amplification

Abstract

An isothermal colorimetric method is described for amplified detection of the CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on (a) target DNA-triggered unlabeled molecular beacon (UMB) termini binding, and (b) exonuclease III (Exo III)-assisted target recycling, and (c) hemin/G-quadruplex (DNAzyme) based signal amplification. The specific binding of target to the G-quadruplex sequence-locked UMB triggers the digestion of Exo III. This, in turn, releases an active G-quadruplex segment and target DNA for successive hybridization and cleavage. The Exo III impellent recycling of targets produces numerous G-quadruplex sequences. These further associate with hemin to form DNAzymes and hence will catalyze HO-mediated oxidation of the chromogenic enzyme substrate ABTS causing the formation of a green colored product. This finding enables a sensitive colorimetric determination of GMO DNA (at an analytical wavelength of 420 nm) at concentrations as low as 0.23 nM. By taking advantage of isothermal incubation, this method does not require sophisticated equipment or complicated syntheses. Analyses can be performed within 90 min. The method also discriminates single base mismatches. In our perception, it has a wide scope in that it may be applied to the detection of many other GMOs. Graphical abstract An isothermal and sensitive colorimetric method is described for amplified detection of CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on target DNA-triggered molecular beacon (UMB) termini-binding and exonuclease III assisted target recycling, and on hemin/G-quadruplex (DNAzyme) signal amplification.

Rapid and Ultrasensitive Fluorescence Sensing Platform Based on Nanometer-Sized Metal–Organic Frameworks for Transgenic CaMV 35S Promoter Detection

“…Nevertheless, this method has some inherent defects, including time-consuming process, easy mismatch and nonspecific amplification, high cost, and expensive equipment. 11,12 In addition to PCR, several other assays are available, including colorimetry, 13 phosphorescence resonance energy transfer (PRET), 14 electrochemistry, 15 surface plasmon resonance (SPR) techniques, 16 and so on.…”

“…Currently, the main method reported for GMO detection is polymerase chain reaction (PCR) − combined with gel electrophoresis. Nevertheless, this method has some inherent defects, including time-consuming process, easy mismatch and nonspecific amplification, high cost, and expensive equipment. , In addition to PCR, several other assays are available, including colorimetry, phosphorescence resonance energy transfer (PRET), electrochemistry, surface plasmon resonance (SPR) techniques, and so on. However, these methods exhibit some disadvantages such as high detection limit, narrow detection range, and complex operation.…”

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

“…In addition to cleaving-DNAzyme, the G-quadruplex-DNAzyme is another type of DNAzyme in which G-rich sequences can fold into a parallel or an antiparallel G-quadruplex in the presence of K + or Pb 2+ . For example, in the presence of hemin, G-quadruplex DNAzyme demonstrates a horseradish peroxidase-mimic activity to catalyze the conversion of a colorless ABTS 2- to a green ABTS ·- , serving as a valuable tool for visual nucleic acid detection [48,49,50].…”

Strand Displacement Amplification Assisted CRISPR-Cas12a Strategy for Colorimetric Analysis of Viral Nucleic Acid

“…Colorimetry is widely applied for biosensing owing to the advantages of easy operation and naked eye visualization. , Also, various colorimetric strategies have been proposed for viral molecular diagnosis. − Nevertheless, the practical application of these strategies is always limited by their low detection sensitivity. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are initially derived from bacteria and archaea and have been explored for molecular diagnosis. − CRISPR-Cas12a can recognize a double-strand DNA (dsDNA) target with a protospacer adjacent motif (PAM) sequence (5′-TTTN-3′) or a single-strand DNA (ssDNA) target and then indiscriminately cleave the ssDNA reporter as the single-strand DNase .…”