ABSTRACT
Agri-food safety has been considered as one of the most important public concerns worldwide. From farm to table, food crops and foods are extremely vulnerable to the contamination by a variety of pollutants from their growth and processing. Moreover, the SARS-CoV-2 detected in the food supply chain during COVID-19 pandemic has posed a greater challenge for rapid and on-site detection of agri-food contaminants in complex and volatile environments. Therefore, the development of rapid, accurate, and on-site detection technologies and portable detection devices is of great importance to ensure the agri-food security. This review comprehensively summarized the recent advances on the construction of CRISPR/Cas systems-based biosensing technologies and their portable detection devices, as well as their promising applications in the field of agri-food safety. First of all, the classification and working principles of CRISPR/Cas systems were introduced. Then, the latest advances on the CRISPR/Cas system-based on-site detection technologies and portable detection devices were also systematically summarized. Most importantly, the state-of-the-art applications of CRISPR/Cas systems-based on-site detection technologies and portable detection devices in the fields of agri-food safety were comprehensively summarized. Impressively, the future opportunities and challenges in this emerging and promising field were proposed. Emerging CRISPR/Cas system-based on-site detection technologies have showed a great potential in the detection of agri-food safety. Impressively, the integration of CRISPR/Cas systems-based biosensing technologies with portable detection devices (e.g., nanopore-based detection devices, lateral flow assay, smartphone-based detection devices, and microfluidic devices) is very promising for the on-site detection of agri-food contaminants. Additionally, CRISPR/Cas system-based biosensing technologies can be further integrated with much more innovative technologies for the development of novel detection platforms to realize the more reliable on-site detection of agri-food safety.
ABSTRACT
Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important. Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b. Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples. Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.
ABSTRACT
Severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic has become a global public health emergency. The detection of SARS-CoV-2 and human enteric pathogens in wastewater can provide an early warning of disease outbreak. Herein, a sensitive, multiplexed, colorimetric detection (termed "SMCD") method was established for pathogen detection in wastewater samples. The SMCD method integrated on-chip nucleic acid extraction, two-stage isothermal amplification, and colorimetric detection on a 3D printed microfluidic chip. The colorimetric signal during nucleic acid amplification was recorded in real-time and analyzed by a programmed smartphone without the need for complicated equipment. By combining two-stage isothermal amplification assay into the integrated microfluidic platform, we detected SARS-CoV-2 and human enteric pathogens with sensitivities of 100 genome equivalent (GE)/mL and 500 colony-forming units (CFU)/mL, respectively, in wastewater within one hour. Additionally, we realized smart, connected, on-site detection with a reporting framework embedded in a portable detection platform, which exhibited potential for rapid spatiotemporal epidemiologic data collection regarding the environmental dynamics, transmission, and persistence of infectious diseases.