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

Abstract

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.

CRISPR/Cas systems combined with DNA nanostructures for biomedical applications

“…43 Qiao's group combined the tetrahedral DNA nanostructure mediated hyperbranched hybridization chain reaction (TDN-hHCR) with the CRISPR/Cas12a system, and established a sensitive detection method for Salmonella by means of immunomagnetic separation. 44 In this system, the surface of gold nanospheres (AuNPs) is modified with antibodies that specifically recognize Salmonella, while also being labeled with biological barcode DNA. As shown in Fig.…”

Agarose Hydrogel-Boosted One-Tube RPA-CRISPR/Cas12a Assay for Robust Point-of-Care Detection of Zoonotic Nematode Anisakis

“…By comparison, agarose hydrogel has more advantages over other additives, as it was not as viscid as glycerol, which was hard to transfer, eliminating the need for manual operations or centrifugation during the reaction. 8 Thus, the agarose-improved RPA-CRISPR/Cas12a assay did not require centrifugation, mixing steps, and control of the reaction process by professionals like other strategies (e.g., photoactivation, microfluidics, and glycerol methods) during the reaction. The one-tube RPA-CRISPR system took 2 h to complete the reaction.…”

“…Nucleic acid amplification testing, for instance, polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR), are frequently used techniques in nucleic acid molecular diagnostics. 8 Nevertheless, these methods require dedicated instruments and sophisticated operation steps, and they could not satisfy the need for point-of-care testing (POCT). Therefore, a detection method that is ultrasensitive, specific, user-friendly, and deliverable to end-users is essential for food safety supervision.…”

“…So far, many methods have been developed for anisakid detection, for example, candling, digestion, and ultraviolet (UV) illumination. , However, the complete destruction of fish has limited the large-scale application of these methods in the food industry. Nucleic acid amplification testing, for instance, polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR), are frequently used techniques in nucleic acid molecular diagnostics . Nevertheless, these methods require dedicated instruments and sophisticated operation steps, and they could not satisfy the need for point-of-care testing (POCT).…”

Broad-Spectrum Detection of Tetracyclines by Riboswitch-Based Cell-Free Expression Biosensing

“…6 For the European Union, the amount of residue acute exposure assessment tolerance for TCs is 9.6 mg•kg −1 for animal species kidney and 0.1 mg•kg −1 for milks. 7 Current methods for the detection and quantification of TCs are mainly based on the combination of liquid chromatography with other methods (e.g., mass spectrometry, and UV detection) and capillary electrophoresis. 8 These analytical methods require large instruments and therefore do not allow for on-site screening tests.…”