Label-Free Sequence-Specific Visualization of LAMP Amplified Salmonella via DNA Machine Produces G-Quadruplex DNAzyme

Abstract

Salmonella is one of four key global causes of diarrhea, and in humans, it is generally contracted through the consumption of contaminated food. It is necessary to develop an accurate, simple, and rapid method to monitor Salmonella in the early phase. Herein, we developed a sequence-specific visualization method based on loop-mediated isothermal amplification (LAMP) for the detection of Salmonella in milk. With restriction endonuclease and nicking endonuclease, amplicons were produced into single-stranded triggers, which further promoted the generation of a G-quadruplex by a DNA machine. The G-quadruplex DNAzyme possesses peroxidase-like activity and catalyzes the color development of 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) as the readouts. The feasibility for real samples analysis was also confirmed with Salmonella spiked milk, and the sensitivity was 800 CFU/mL when observed with the naked eye. Using this method, the detection of Salmonella in milk can be completed within 1.5 h. Without the involvement of any sophisticated instrument, this specific colorimetric method can be a useful tool in resource-limited areas.

Recent progress on DNAzyme-based biosensors for pathogen detection

“…To address this constraint, the conjunction of nucleic acid amplification technologies and HGD has been employed to achieve substantial signal amplification, elevating the detection capabilities by orders of magnitude. Various nucleic acid amplification techniques are being integrated with HGD for detection purposes, such as Polymerase Chain Reaction (PCR), 32–37 Loop-Mediated Isothermal Amplification (LAMP), 38–44 and Recombinase Polymerase Amplification (RPA). 45–48 These methods are able to amplify the signal to be detected.…”

“…40–42 Another strategy based on LAMP is to cleave the amplicon to generate the ssDNA product, which can amplify the sequence of the G-quadruplex. 43 They added a restriction endonuclease recognition site to the primers. The cleavage fragment was used as a primer for amplification in combination with another template strand.…”

Label-Free Biosensor

“…Six articles are included to show the development and applications of novel label-free biosensors. Xu et al [1] developed a sequence-specific visualization method based on loop-mediated isothermal amplification for the detection of Salmonella-one of four key global causes of diarrhea-in milk. This method does not involve any sophisticated instrument; thus, it may be a useful tool in resource-limited areas.…”