Dual-Functional Tetrahedron Multivalent Aptamer Assisted Amplification-Free CRISPR/Cas12a Assay for Sensitive Detection of Salmonella.

Salmonellosis continues to impose a significant economic burden globally. Rapid and sensitive detection of Salmonella is crucial to preventing the outbreaks of foodborne illnesses, yet it remains a formidable challenge. Herein, a dual-functional tetrahedron multivalent aptamer assisted amplification-free CRISPR/Cas12a assay was developed for Salmonella detection. In the system, the aptamer was programmatically assembled on the tetrahedral DNA nanostructure to fabricate a multivalent aptamer (TDN-multiApt), which displayed a 3.5-fold enhanced avidity over the monovalent aptamer and possessed four CRISPR/Cas12a targeting fragments to amplify signal. Therefore, TDN-multiApt could directly activate Cas12a to achieve the second signal amplification without any nucleic acid amplification. By virtue of the synergism of high avidity and cascaded signal amplifications, the proposed method allowed the ultrasensitive detection of Salmonella as low as 7 cfu mL-1. Meanwhile, this novel platform also exhibited excellent specificity against target bacteria and performed well in the detection of various samples, indicating its potential application in real samples.

A Colorimetric/Fluorescent Dual-Mode Aptasensor for Salmonella Based on the Magnetic Separation of Aptamers and a DNA-Nanotriangle Programmed Multivalent Aptamer.

Salmonella infection has emerged as a global health threat, causing death, disability, and socioeconomic disruption worldwide. The rapid and sensitive detection of Salmonella is of great significance in guaranteeing food safety. Herein, we developed a colorimetric/fluorescent dual-mode method based on a DNA-nanotriangle programmed multivalent aptamer for the sensitive detection of Salmonella. In this system, aptamers are precisely controlled and assembled on a DNA nanotriangle structure to fabricate a multivalent aptamer (NTri-Multi-Apt) with enhanced binding affinity and specificity toward Salmonella. The NTri-Multi-Apt was designed to carry many streptavidin-HRPs for colorimetric read-outs and a large load of Sybr green I in the dsDNA scaffold for the output of a fluorescent signal. Therefore, combined with the magnetic separation of aptamers and the prefabricated NTri-Multi-Apt, the dual-mode approach achieved simple and sensitive detection, with LODs of 316 and 60 CFU/mL for colorimetric and fluorescent detection, respectively. Notably, the fluorescent mode provided a self-calibrated and fivefold-improved sensitivity over colorimetric detection. Systematic results also revealed that the proposed dual-mode method exhibited high specificity and applicability for milk, egg white, and chicken meat samples, serving as a promising tool for real bacterial sample testing. As a result, the innovative dual-mode detection method showed new insights for the detection of other pathogens.

Sensitive Detection of Salmonella Based on CRISPR-Cas12a and the Tetrahedral DNA Nanostructure-Mediated Hyperbranched Hybridization Chain Reaction.

Salmonella severely threatens global human health and causes financial burden. The ability to sensitively detect Salmonella in food samples is highly valuable but remains a challenge. Herein, a sensitive detection method for Salmonella was developed by coupling immunomagnetic separation with the CRISPR-Cas12a system and the tetrahedral DNA nanostructure-mediated hyperbranched hybridization chain reaction (TDN-hHCR). In the detection system, the target Salmonella was immunomagnetically separated and labeled with bio-barcode DNA-modified gold nanoparticles (AuNPs), which could transfer and magnify the signal of a bacterial cell into numerous bio-barcode DNA molecules. Afterward, the bio-barcode DNA can trigger the trans-cleavage activity of CRISPR-Cas12a to inhibit the process of the TDN-hHCR to generate a fluorescence readout. Due to the high immunomagnetic separation efficiency and the effective signal amplification of CRISPR-Cas12a and the TDN-hHCR, Salmonella as low as 8 CFU/mL could be easily detected. Meanwhile, this has been applied for practical use and showed the capability to detect 17 and 25 CFU/mL in spiked milk and egg white, respectively, indicating its potential application in real samples.