SERS-based CRISPR/Cas assay on microfluidic paper analytical devices for supersensitive detection of pathogenic bacteria in foods.

Novel non-nucleic acid targets detection strategies based on CRISPR/Cas toolboxes: A review.

In the context of the current pandemic caused by the novel coronavirus, molecular detection is not limited to the clinical laboratory, but also faces the challenge of the complex and variable real-time detection fields. A series of novel coronavirus events were detected in the process of food cold chain packaging and transportation, making the application of molecular diagnosis in food processing, packaging, transportation, and other links urgent. There is an urgent need for a rapid detection technology that can adapt to the diversity and complexity of food safety. This review introduces a new molecular diagnostic technology-biosensor analysis technology based on CRISPR-Cas12a. Systematic clarification of its development process and detection principles. It summarizes and systematically organizes its applications in viruses, food-borne pathogenic bacteria, small molecule detection, etc. In the past four years, which provides a brand-new and comprehensive solution for food detection. Finally, this article puts forward the challenges and the prospects for food safety. The novel coronavirus hazards infiltrated every step of the food industry, from processing to packaging to transportation. The biosensor analytical technology based on CRISPR-Cas12a has great potential in the qualitative and quantitative analysis of infectious pathogens. CRISPR-Cas12a can effectively identify the presence of the specific nucleic acid targets and the small changes in sequences, which is particularly important for nucleic acid identification and pathogen detection. In addition, the CRISPR-Cas12a method can be adjusted and reconfigured within days to detect other viruses, providing equipment for nucleic acid diagnostics in the field of food safety. The future work will focus on the development of portable microfluidic devices for multiple detection. Shao et al. employed physical separation methods to separate Cas proteins in different microfluidic channels to achieve multiple detection, and each channel simultaneously detected different targets by adding crRNA with different spacer sequences. Although CRISPR-Cas12a technology has outstanding advantages in detection, there are several technical barriers in the transformation from emerging technologies to practical applications. The newly developed CRISPR-Cas12a-based applications and methods promote the development of numerous diagnostic and detection solutions, and have great potential in medical diagnosis, environmental monitoring, and especially food detection. 

https://doi.org/10.1016/j.tifs.2022.02.030

CRISPR/Cas12a-based technology: A powerful tool for biosensing in food safety

Food safety is one of the biggest public issues occurring around the world. Microbiological, chemical, and physical hazards can lead to food safety issues, which may occur at all stages of the supply chain. In order to tackle food safety issues and safeguard consumer health, rapid, accurate, specific, and field-deployable detection methods meeting diverse requirements are one of the imperative measures for food safety assurance. CRISPR-Cas system, a newly emerging technology, has been successfully repurposed in biosensing and has demonstrated huge potential to establish conceptually novel detection methods with high sensitivity and specificity. This review focuses on CRISPR-Cas-based detection and its current status and huge potential specifically for food safety inspection. We firstly illustrate the pending problems in food safety and summarize the popular detection methods. We then describe the potential applications of CRISPR-Cas-based detection in food safety inspection. Finally, the challenges and futuristic opportunities are proposed and discussed. Generally speaking, the current food safety detection methods are still unsatisfactory in some ways such as being time-consuming, displaying unmet sensitivity and specificity standards, and there is a comparative paucity of multiplexed testing and POCT. Recent studies have shown that CRISPR-Cas-based biosensing is an innovative and fast-expanding technology, which could make up for the shortcomings of the existing methods or even replace them. To sum up, the implementation of CRISPR-Cas and the integration of CRISPR-Cas with other techniques is promising and desirable, which is expected to provide "customized" and "smart" detection methods for food safety inspection in the coming future.

CRISPR-Cas-based detection for food safety problems: Current status, challenges, and opportunities.

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform

A smartphone-based visual biosensor for CRISPR-Cas powered SARS-CoV-2 diagnostics.

Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenient. In this study, we developed a G-quadruplex-based CRISPR-Cas12a bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella specific invA gene activated the trans-cleavage activity of Cas12a and triggered CRISPR-Cas12a based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K+. This DNAzyme could catalyze the TMB-H2O2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity and on-site capability.

Si Chen

Papers

A smartphone-based visual biosensor for CRISPR-Cas powered SARS-CoV-2 diagnostics.

Ultrasensitive pathogenic bacteria detection by a smartphone-read G-quadruplex-based CRISPR-Cas12a bioassay