A new simplified sequence-dependent loop-mediated isothermal amplification (LAMP) detection method.

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection method, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the 5' end of the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10 min faster than several other probe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection.

A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane.

Nucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microfluidic chips, making it difficult to popularize in the point-of-care detection. Paper is an inexpensive and readily available material. In this study, we propose a simple and cost-effective paper membrane-based digital loop-mediated isothermal amplification (LAMP) method for nucleic acid quantification. In the presence of DNA fluorescence dyes, the high background signals will cover up the amplicons-formed bright spots. To reduce the background fluorescence signals, a quencher-fluorophore duplex was introduced in LAMP primers to replace non-specific fluorescence dyes. After that, the amplicons-formed spots on the paper membrane can be observed; thus, the target DNA can be quantified by counting the spots. Take Vibrio parahaemolyticus DNA detection as an instance, a good linear relationship is obtained between the light spots and the copy numbers of DNA. The paper membrane-based digital LAMP detection can detect 100 copies target DNA per reaction within 30 min. Overall, the proposed nucleic acid quantification method has the advantages of a simple workflow, short sample-in and answer-out time, low cost, and high signal-to-noise, which is promising for application in resourced limited areas.

Toripalimab Plus Chemotherapy for Recurrent or Metastatic Nasopharyngeal Carcinoma: The JUPITER-02 Randomized Clinical Trial.

Importance: There are currently no therapies approved by the US Food and Drug Administration for nasopharyngeal carcinoma (NPC). Gemcitabine-cisplatin is the current standard of care for the first-line treatment of recurrent or metastatic NPC (RM-NPC). Objective: To determine whether toripalimab in combination with gemcitabine-cisplatin will significantly improve progression-free survival and overall survival as first-line treatment for RM-NPC, compared with gemcitabine-cisplatin alone. Design, Setting, and Participants: JUPITER-02 is an international, multicenter, randomized, double-blind phase 3 study conducted in NPC-endemic regions, including mainland China, Taiwan, and Singapore. From November 10, 2018, to October 20, 2019, 289 patients with RM-NPC with no prior systemic chemotherapy in the RM setting were enrolled from 35 participating centers. Interventions: Patients were randomized (1:1) to receive toripalimab (240 mg [n = 146]) or placebo (n = 143) in combination with gemcitabine-cisplatin for up to 6 cycles, followed by maintenance with toripalimab or placebo until disease progression, intolerable toxicity, or completion of 2 years of treatment. Main Outcome: Progression-free survival as assessed by a blinded independent central review. Secondary end points included objective response rate, overall survival, progression-free survival assessed by investigator, duration of response, and safety. Results: Among the 289 patients enrolled (median age, 46 [IQR, 38-53 years; 17% female), at the final progression-free survival analysis, toripalimab treatment had a significantly longer progression-free survival than placebo (median, 21.4 vs 8.2 months; HR, 0.52 [95% CI, 0.37-0.73]). With a median survival follow-up of 36.0 months, a significant improvement in overall survival was identified with toripalimab over placebo (hazard ratio [HR], 0.63 [95% CI, 0.45-0.89]; 2-sided P = .008). The median overall survival was not reached in the toripalimab group, while it was 33.7 months in the placebo group. A consistent effect on overall survival, favoring toripalimab, was found in subgroups with high and low PD-L1 (programmed death-ligand 1) expression. The incidence of all adverse events, grade 3 or greater adverse events, and fatal adverse events were similar between the 2 groups. However, adverse events leading to discontinuation of toripalimab or placebo (11.6% vs 4.9%), immune-related adverse events (54.1% vs 21.7%), and grade 3 or greater immune-related adverse events (9.6% vs 1.4%) were more frequent in the toripalimab group. Conclusions and Relevance: The addition of toripalimab to chemotherapy as first-line treatment for RM-NPC provided statistically significant and clinically meaningful progression-free survival and overall survival benefits compared with chemotherapy alone, with a manageable safety profile. These findings support the use of toripalimab plus gemcitabine-cisplatin as the new standard of care for this patient population. Trial Registration: ClinicalTrials.gov Identifier: NCT03581786.

Cooperative Rh-O5/Ni(Fe) Site for Efficient Biomass Upgrading Coupled with H2 Production.

Designing efficient and durable bifunctional catalysts for 5-hydroxymethylfurfural (HMF) oxidation reaction (HMFOR) and hydrogen evolution reaction (HER) is desirable for the co-production of biomass-upgraded chemicals and sustainable hydrogen, which is limited by the competitive adsorption of hydroxyl species (OHads) and HMF molecules. Here, we report a class of Rh-O5/Ni(Fe) atomic site on nanoporous mesh-type layered double hydroxides with atomic-scale cooperative adsorption centers for highly active and stable alkaline HMFOR and HER catalysis. A low cell voltage of 1.48 V is required to achieve 100 mA cm-2 in an integrated electrolysis system along with excellent stability (>100 h). Operando infrared and X-ray absorption spectroscopic probes unveil that HMF molecules are selectively adsorbed and activated over the single-atom Rh sites and oxidized by in situ-formed electrophilic OHads species on neighboring Ni sites. Theoretical studies further demonstrate that the strong d-d orbital coupling interactions between atomic-level Rh and surrounding Ni atoms in the special Rh-O5/Ni(Fe) structure can greatly facilitate surface electronic exchange-and-transfer capabilities with the adsorbates (OHads and HMF molecules) and intermediates for efficient HMFOR and HER. We also reveal that the Fe sites in Rh-O5/Ni(Fe) structure can promote the electrocatalytic stability of the catalyst. Our findings provide new insights into catalyst design for complex reactions involving competitive adsorptions of multiple intermediates.

A Designed Vessel Using Dissolvable Polyvinyl Alcohol Membrane as Automatic Valve to Couple LAMP with CRISPR/Cas12a System for Visual Detection.

A rapid and intuitive method for detecting Vibrio parahaemolyticus (VP) was established by a designed reaction vessel which coupled CRISPR/Cas12a with loop-mediated isothermal nucleic acid amplification (LAMP). There were two spaces in the vessel-holding LAMP reaction solution and CRISPR reaction solution, respectively, which were separated with a polyvinyl alcohol (PVA) membrane. The PVA membrane could be dissolved with a water solution. The thermolabile hemolysin (TLH) gene of VP was employed as the detection target. After the target sequence of the TLH gene was amplified with LAMP, the PVA membrane would be dissolved and the CRISPR reaction solution mixed with the LAMP reaction solution. In this way, amplicons could be detected with CRISPR/Cas12a in the reaction vessel. The fluorescent signals produced by the positive samples were clearly identified by the naked eye under a UV light, while the negative samples were dark. The whole detection procedure could be finished within 35 min with a detection limit of 100 copies/µL. The designed reaction vessel is easy to produce and can effectively prevent contamination due to the opening of the reaction vessel after the LAMP reaction. Thus, it will have the potential to provide a new solution for rapid detection in the field.

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.

Atomically Dispersed CoN3 C1 -TeN1 C3 Diatomic Sites Anchored in N-Doped Carbon as Efficient Bifunctional Catalyst for Synergistic Electrocatalytic Hydrogen Evolution and Oxygen Reduction.

A encapsulation-adsorption-pyrolysis strategy for the construction of atomically dispersed Co-Te diatomic sites (DASs) that are anchored in N-doped carbon is reported as an efficient bifunctional catalyst for electrocatalytic hydrogen evolution reaction (HER) and oxygen reduction reaction (ORR). The as-constructed catalyst shows the stable CoN3 C1 -TeN1 C3 coordination structure before and after HER and ORR. The *OOH/*H intermediate species are captured by in situ Raman and in situ attenuated total reflectance-surface enhanced infrared absorption spectroscopy, indicating that the reactant O2 /H2 O molecule has a strong interaction with the Co site, revealing that Coδ+ is an effective active site. Theoretical calculations show that the Coδ+ has adsorption-activation function and the neighboring Teδ+ acts as an electron donor adjusting the electronic structure of Coδ+ , promoting the dissociation of H2 O molecules and the adsorption of H and oxygen-containing intermediates in HER and ORR. In the meanwhile, the nearest C atom around Co also profoundly affects the adsorption of H atoms. This results in the weakening of the OH adsorption and enhancement of H adsorption, as well as the more stable water molecule dissociation transition state, thus significantly boosting ORR and HER performance.

Synergetic Function of the Single-Atom Ru-N4 Site and Ru Nanoparticles for Hydrogen Production in a Wide pH Range and Seawater Electrolysis.

Hydrogen production by water splitting and seawater electrolysis is a promising alternative to develop clean hydrogen energy. The construction of high-efficiency and durable electrocatalysts for the hydrogen evolution reaction (HER) in a wide pH range and seawater is critical to overcoming the sluggish kinetic process. Herein, we develop an efficient catalytic material composed of a single-atom Ru-N4 site and Ru nanoparticles anchored on nitrogen-doped carbon (Ru1+NPs/N-C) through the coordination-pyrolysis strategy of the melamine formaldehyde resin. The Ru1+NPs/N-C catalyst shows outstanding HER activity with the smallest overpotentials, the lowest Tafel slopes, the highest mass activity and turnover frequency, as well as excellent stability in both acidic and alkaline media. Moreover, Ru1+NPs/N-C shows comparable hydrogen production performance and a higher faradic efficiency to 20% Pt/C in natural seawater and artificial simulated seawater. Theoretical calculations demonstrate that the strong synergistic effects between the Ru-N4 site and Ru nanoparticles modify the electronic structure to accelerate the HER kinetics. Ru nanoparticles can effectively realize dissociation of H2O to generate adsorbed hydrogen and also promote the single-atom Ru-N4 site to combine adsorbed hydrogen to H2 and desorption. This work provides a new perspective for designing high-efficiency hydrogen production electrocatalysts for large-scale seawater electrolysis.

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.

Toripalimab or placebo plus chemotherapy as first-line treatment in advanced nasopharyngeal carcinoma: a multicenter randomized phase 3 trial.

Gemcitabine-cisplatin (GP) chemotherapy is the standard first-line systemic treatment for recurrent or metastatic nasopharyngeal carcinoma (RM-NPC). In this international, double-blind, phase 3 trial (ClinicalTrials.gov identifier: NCT03581786), 289 patients with RM-NPC and no previous chemotherapy for recurrent or metastatic disease were randomized (1/1) to receive either toripalimab, a monoclonal antibody against human programmed death-1 (PD-1), or placebo in combination with GP every 3 weeks for up to six cycles, followed by monotherapy with toripalimab or placebo. The primary endpoint was progression-free survival (PFS) as assessed by a blinded independent review committee according to RECIST v.1.1. At the prespecified interim PFS analysis, a significant improvement in PFS was detected in the toripalimab arm compared to the placebo arm: median PFS of 11.7 versus 8.0 months, hazard ratio (HR) = 0.52 (95% confidence interval (CI): 0.36-0.74), P = 0.0003. An improvement in PFS was observed across key subgroups, including PD-L1 expression. As of 18 February 2021, a 40% reduction in risk of death was observed in the toripalimab arm compared to the placebo arm (HR = 0.603 (95% CI: 0.364-0.997)). The incidence of grade ≥3 adverse events (AEs) (89.0 versus 89.5%), AEs leading to discontinuation of toripalimab/placebo (7.5 versus 4.9%) and fatal AEs (2.7 versus 2.8%) was similar between the two arms; however, immune-related AEs (39.7 versus 18.9%) and grade ≥3 infusion reactions (7.5 versus 0.7%) were more frequent in the toripalimab arm. In conclusion, the addition of toripalimab to GP chemotherapy as a first-line treatment for patients with RM-NPC provided superior PFS compared to GP alone, and with a manageable safety profile.

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.

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.