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.

A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern.

BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).

Dual-detection fluorescent immunochromatographic assay for quantitative detection of SARS-CoV-2 spike RBD-ACE2 blocking neutralizing antibody.

The global effort against the COVID-19 pandemic dictates that routine quantitative detection of SARS-CoV-2 neutralizing antibodies is vital for assessing immunity following periodic revaccination against new viral variants. Here, we report a dual-detection fluorescent immunochromatographic assay (DFIA), with a built-in self-calibration process, that enables rapid quantitative detection of neutralizing antibodies that block binding between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2). Thus, this assay is based on the inhibition of binding between ACE2 and the RBD of the SARS-CoV-2 spike protein by neutralizing antibodies, and the affinity of anti-human immunoglobulins for these neutralizing antibodies. Our self-calibrating DFIA shows improved precision and sensitivity with a wider dynamic linear range, due to the incorporation of a ratiometric algorithm of two-reverse linkage signals responding to an analyte. This was evident by the fact that no positive results (0/14) were observed in verified negative samples, while 22 positives were detected in 23 samples from verified convalescent plasma. A comparative analysis of the ability to detect neutralizing antibodies in 266 clinical serum samples including those from vaccine recipients, indicated that the overall percent agreement between DFIA and the commercial ELISA kit was 90.98%. Thus, the proposed DFIA provides a more reliable and accurate rapid test for detecting SARS-CoV-2 infections and vaccinations in the community. Therefore, the DFIA based strategy for detecting biomarkers, which uses a ratiometric algorithm based on affinity and inhibition reactions, may be applied to improve the performance of immunochromatographic assays.

Carrying out pseudo dual nucleic acid detection from sample to visual result in a polypropylene bag with CRISPR/Cas12a.

Amplification-based nucleic acid detection is widely employed in food safety, medical diagnosis and environment monitoring. However, conventional nucleic acid analysis has to be carried out in laboratories because of requiring expensive instruments and trained personnel. If people could do nucleic acid detection at home by themselves, the application of nucleic acid detection would be greatly accelerated. We herein reported a polypropylene (PP) bag-based method for convenient detection of nucleic acids in the oil-sealed space. The PP bag has three chambers which are responsible for lysis, washing and amplification/detection, respectively. After adding sample, nucleic acids are adsorbed on magnetic particles (MPs) and moved into these three chambers successively through immiscible oil channel by an external magnet. Combined with isothermal amplification, the PP bag can be incubated in a water bath or milk warmer and acted as a reaction tube. With highly specific CRISPR technology, Salmonella typhimurium (St) and SARS-CoV-2 can be visually detected in these PP bags within 1 h, indicating its potential household application. To further improve the reliability of nucleic acid testing at home, a logic decision method is introduced by detecting both target and endogenous reference gene. Positive/negative/invalid detection result can be obtained by chronologically adding the CRISPR reagents of target and endogenous reference gene. We anticipate that this PP bag can provide a novel toolkit for nucleic acid detection in people's daily life.