CRISPR/Cas12a-Assisted Dual Visualized Detection of SARS-CoV-2 on Frozen Shrimps.

Given the possibility that food contaminated with SARS-CoV-2 might become an infection source, there is an urgent need for us to develop a rapid and accurate nucleic acid detection method for SARS-CoV-2 in food to ensure food safety. Here, we propose a sensitive, specific, and reliable molecular detection method for SARS-CoV-2. It has a mechanism to control amplicon contamination. Swabs from spiked frozen shrimps were used as detection samples, which were processed by heating at 95 °C for 30 s. These preprocessed samples served as the templates for subsequent amplification. A colorimetric LAMP reaction was carried out to amplify both the SARS-CoV-2 target and the MS2 phage simultaneously in one tube. MS2 phage was detected by colorimetric LAMP as the internal control, while SARS-CoV-2 was detected with a CRISPR/Cas12a system. The fluorescence results could be visually detected with an ultraviolet lamp. Meanwhile, uracil was incorporated during the LAMP reaction to provide an amplicon contamination proof mechanism. This test could detect as low as 20 copies of SARS-CoV-2 in one reaction. Additionally, the detection could be finished in 45 min. The test only needs a heating block and an ultraviolet lamp, which shows the potential for field detection.

Integrated slip valve-assisted fluidic chip coupling with CRISPR/Cas12a system for nucleic acid analysis.

Currently, some on-site nucleic acid detection platforms have been developed. However, these platforms still need to be improved in device integration and multiple detection capability. In this work, an integrated dual nucleic acid analysis platform was developed by slip valve-assisted fluidic chip coupled with CRISPR/Cas12a system. All the reagents, including nucleic acid extraction, air-dried loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a detection reagents, were preloaded on the fluidic chip. Liquids transfer and stirring could be controlled by a slip valve and a syringe. By combining duplex LAMP reaction with two CRISPR detection units, CRISPR/Cas12a-based dual nucleic acid analysis was successfully constructed. Benefiting from high-quality nucleic acid extraction on the chip, as low as 30 copies/reaction of Vibrio parahaemolyticus (V. parahaemolyticus) and 20 copies/reaction of Salmonella typhimurium (S. typhimurium) could be simultaneously detected. Detection results could be observed by the naked eye under a portable ultraviolet lamp. The whole detection procedure was finished within 60 min. This method with integrated nucleic acid analysis, dual detection capability and fluorescence visualized results provides a new solution for on-site nucleic acid analysis.

Microfluidics: the propellant of CRISPR-based nucleic acid detection.

Since the discovery of collateral cleavage activity, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have become the new generation of nucleic acid detection tools. However, their widespread application remains limited. A pre-amplification step is required to improve the sensitivity of CRISPR systems, complicating the operating procedure and limiting quantitative precision. In addition, nonspecific collateral cleavage activity makes it difficult to realize multiplex detection in a one-pot CRISPR reaction with a single Cas protein. Microfluidics, which can transfer nucleic acid analysis process to a chip, has the advantages of miniaturization, integration, and automation. Microfluidics coupled with CRISPR systems improves the detection ability of CRISPR, enabling fast, high-throughput, integrated, multiplex, and digital detection, which results in the further popularization of CRISPR for a range of scenarios.

Multiple crRNAs-assisted CRISPR/Cas12a assay targeting cytochrome b gene for amplification-free detection of meat adulteration.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have been widely applied in nucleic acid analysis for the high specificity. Coupled with pre-amplification steps, the sensitivity of CRISPR-based detection is greatly improved. However, an extra pre-amplification step not only complicates the detection procedures but may also cause aerosol contaminations in the process of transferring amplified solution into CRISPR system. In this study, we demonstrate that combination of multiple crRNAs in CRISPR/Cas12a system can enhance the detection sensitivity. Based on it, we establish a multiple crRNAs enhanced CRISPR (meCRISPR) method and apply it to meat adulteration identification. Take cytochrome b (Cyt b) gene as a target, meCRISPR method can directly detect as low as 1.13 ng/µL extracted pork DNA and 5% (w/w) pork contamination in pork and beef meat mixtures. There is no cross-reaction with extracted chicken, beef, duck and fish DNA. meCRISPR reaction is incubated at an isothermal temperature, and the detection process can be completed in a designed portable apparatus with a heat block, a light emitting diode and filters. For the simplicity, specificity and sufficient sensitivity of meCRISPR method, it will have great prospects in species identification, food adulteration, and genetically modified food detection.

Dipstick-based rapid nucleic acids purification and CRISPR/Cas12a-mediated isothermal amplification for visual detection of African swine fever virus.

African swine fever virus (ASFV) can cause highly contagious and fatal disease among domestic pigs, resulting in considerable economic losses for swine breeders. There is a strong demand for accurate, rapid, and simple detection methods especially for on-site application. Nucleic acid testing is the most commonly used method for ASFVdetection. However, traditional nucleic acid purification step is time- and labor-consuming. The nucleic acid purification, amplification and amplicons detection rely on laboratory settings which limits the on-site detection. Here, we proposed a simple and cost-effective detection method that utilized filter paper to purify nucleic acids from swine blood and employed CRISPR/Cas12a-mediated loop-mediated isothermal amplification (LAMP) reaction to detect ASFV. The filter paper which was made into dipsticks could effectively purify nucleic acids from whole blood in 2 min. This simple and low-cost purification method avoided multiple pipetting steps and potential amplification inhibitors (e.g., ethanol) that were generally used in traditional nucleic acids extraction processes. After nucleic acid purification, the lyophilized LAMP reagent dissolved by elution solution was employed to perform isothermal amplification reaction on a portable heating block. The CRISPR/Cas12a system was designed to specifically detect amplicons. Assisted by a portable homemade device, the fluorescent signals produced by positive samples could be observed by the naked eye, while negative samples remained colorless. The whole detection procedure could be finished within 50 min with a detection limit of one copies/µL. This established method provided a novel strategy for rapid visualized detection and showed great potential for on-site application.

Advances in amplification-free detection of nucleic acid: CRISPR/Cas system as a powerful tool.

Amplification technologies such as polymerase chain reaction (PCR) play an important role in nucleic acid detection. However, they require bulky and sophisticated thermal cycling instrument, as well as are prone to get false-positive results due to amplicon contamination. Currently, CRISPR/Cas system has become an increasingly popular diagnostic tool for nucleic acid with the discovery of its trans-cleavage activity which can degrade single-stranded DNA or RNA at a very high turnover rate. This inherent signal amplification capability allows CRISPR/Cas system to detect unamplified nucleic acids. Here, we reviewed the recent advances of CRISPR-based amplification-free methods for nucleic acid detection. With the assistance of various signal enhancement strategies, the detection sensitivity could be comparable to that of amplification-based methods. We then presented the pros and cons of these methods. And the subsistent challenges including sample preparation, off-target effect, sequences limit, quantitative and multiplex detection were further discussed in this review. It is probable for CRISPR-powered detection methods to pave the road for rapid, cheap, highly sensitive and specific on-site detection without amplification.

Applying CRISPR/Cas system as a signal enhancer for DNAzyme-based lead ion detection.

Heavy metal lead accumulation in the environment pollutes the ecology systems and further threatens the human health. It is necessary to develop a sensitive method to detect it. Here, we propose a highly sensitive lead detection method by combining DNAzyme and CRISPR system. Once the lead ion is recognized, the substrate chain of DNAzyme is cleaved to produce single strand DNA. The produced single strand DNA can be detected by Cas protein/guide RNA complex and further trigger the collateral cleavage effect of CRISPR system, which can indiscriminately cut short single strand DNA reporters. By this way, the detection signals can be greatly amplified. This method can detect lead ions as low as 0.48 nM. The sensitivity is higher than the DNAzyme method. Furthermore, the portable 3D printing device is designed to observe the fluorescent signals so the end-point detection results can be visualized by the naked eyes. The entire detection process can avoid using bulky and expensive instruments, which can promote on-site lead ion detection.

Rotary Valve-Assisted Fluidic System Coupling with CRISPR/Cas12a for Fully Integrated Nucleic Acid Detection.

Of late, many nucleic acid analysis platforms have been established, but there is still room for constructing integrated nucleic acid detection systems with high nucleic acid extraction efficiency, low detection cost, and convenient operation. In this work, a simple rotary valve-assisted fluidic chip coupling with CRISPR/Cas12a was established to achieve fully integrated nucleic acid detection. All of the detection reagents were prestored on the fluidic chip. With the aid of the rotary valve and syringe, the liquid flow and stirring can be precisely controlled. The nucleic acid extraction, loop-mediated isothermal amplification (LAMP) reaction, and CRISPR detection could be completed in 80 min. A clean reservoir and an air reservoir on the fluidic chip were designed to effectively remove the remaining ethanol. With Vibrio parahaemolyticus as the targets, the detection sensitivity of the fluidic chip could reach 3.1 × 101 copies of target DNA per reaction. A positive sample could be sensitively detected by CRISPR/Cas12a to produce a green fluorescent signal, while a negative sample generated no fluorescent signal. Further, the fluidic chip was successfully applied for detection of spiked shrimp samples, which showed the same detection sensitivity. A great feasibility for real-sample detection was showed by the fluidic chip. The proposed detection platform did not need expensive centrifugal instruments or pumps, which displayed its potential to become a powerful tool for food safety analysis and clinical diagnostics, especially in the resource-limited areas.

A reversible valve-assisted chip coupling with integrated sample treatment and CRISPR/Cas12a for visual detection of Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is regarded as a major cause of seafood-associated illnesses, which has aroused widespread public concern. Here, a rapid and convenient detection method for V. parahaemolyticus detection was established by a reversible valve-assisted chip coupling with CRISPR/Cas12a. With optimized lysis buffer, loop mediated isothermal amplification (LAMP) reagents and CRISPR reagents, the whole detection process from sampling to results could be finished within 50 min. The structure of chip was simple and the cost was low. By relying on three reversible rotary valves and the rotation direction-dependent Coriolis pseudo force, the flow order of liquid and the direction of liquid flow could be precisely controlled. The LAMP amplicons were specifically and sensitively identified by CRISPR/Cas12a. Positive amplification would produce green fluorescent signal while negative amplification generated no fluorescent signal, which could be clearly distinguished by the naked eye. With 600 µL of samples processed, the limit of detection (LOD) for both pure cultured V. parahaemolyticus or spiked shrimp samples could achieve 30 copies/reaction. These illustrated the established method displayed great feasibility for real samples detection. In the future, the chip could also combine with other amplification reactions, like PCR or recombinase polymerase amplification reaction (RPA), to conduct detection by changing the corresponding lyophilized amplification reagents. Overall, the proposed detection platform displays great potential for food safety analysis and clinical diagnostics, especially in resource-limited areas.

CRISPR/Cas12a-Based Versatile Method for Checking Quantitative Polymerase Chain Reaction Samples with Cycles of Threshold Values in the Gray Zone.

Quantitative polymerase chain reaction (qPCR) is widely applied in foodborne pathogen detection and diagnosis. According to the cycles of threshold (Ct) values of qPCR testing, samples are judged as positive or negative. However, samples with Ct values in the gray zone are classified as "possibly positive" and required to be tested again. Repetitive qPCR may not eliminate the uncertain results but increase the workload of detection. CRISPR/Cas12a can specifically recognize the nucleic acid of the nM level and then indiscriminately slash the single-strand DNA with multiple turnovers. In this way, the detection signals can be greatly amplified. Here, we propose a CRISPR-based checking method to solve gray zone problems. After qPCR testing, the screening gray zone samples can be successfully checked by the CRISPR/Cas12a method. Furthermore, to conduct CRISPR reaction assay more conveniently and prevent possible aerosol contamination in the operational process, a gray zone checking cassette is designed. African swine fever virus (ASFV) is selected as an example to demonstrate the feasibility of the CRISPR-based checking method. Of 28 real swine blood samples, 6 ASFV qPCR gray zone samples are successfully checked. The CRISPR-based checking method provides a novel solution to eliminate gray zone sample problems with no additional effects on the PCR, which is operable and applicable in practical detection. The entire process can be completed within 10-15 min. This method will be a good supplementary and assistance for qPCR-based detection, especially in the diagnosis of diseases such as COVID-19.