One-tube nested PCR mediated CRISPR-Cas12a: A novel performance-enhanced approach for ultrasensitive fluorescent visual screening of Salmonella

One-tube nested PCR mediated CRISPR-Cas12a: A novel performance-enhanced approach for ultrasensitive fluorescent visual screening of Salmonella | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article One-tube nested PCR mediated CRISPR-Cas12a: A novel performance-enhanced approach for ultrasensitive fluorescent visual screening of Salmonella Qingli Yang, Noor Fatima, Xiaofeng Yu, Yubo Peng, Qi Chen, Dexing Zeng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7534316/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Salmonella is one of the most prevalent and highly transmissible food-borne pathogens, making rapid and accurate screening essential for safeguarding human health and ensuring food safety. This study introduces a one-tube nested PCR mediated CRISPR-Cas12a for ultrasensitive visual screening of Salmonella spp. using fluorescent lateral flow strip. By leveraging the simultaneous dual-segment amplification capability of the designed one-tube nested PCR and the collateral activated trans -cleavage activity of CRISPR-Cas12a, the method achieves a detection limit of ‌10 1 CFU/mL‌, with no cross-reactivity against other common food-borne pathogens. This approach employs the fluorophore-labeled DNA reporters that are cleaved by activated Cas12a, allowing for rapid and on-site visualization of detection results. Validation in different food matrices yields satisfactory results, demonstrating robustness against matrix interference. Comparative analysis revealed a ‌10-fold sensitivity improvement‌ over traditional single-primer PCR protocols, attributed to the dual amplification efficiency of designed one-tube nested PCR and the collateral activated cleavage specificity of CRISPR-Cas12a. The portability, rapid visual readout, and ultrasensitive performance of the method enable real-time, on-site screening of Salmonella in diverse food supply chains, even in resource-limited settings. Its high specificity, robustness against matrix effects, and minimal equipment requirements make it a transformative, user-friendly tool for enhancing global food safety surveillance and preventing outbreaks. Salmonella one-tube nested PCR simultaneous amplification signal enhancement CRISPR/Cas lateral flow strip visual screening Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Microbial contamination could occur during the growth, harvest, preparation, storage, processing, and distribution of food [ 1 ] . Foodborne pathogens can cause illness or even death of consumers [ 2 ] . Diseases caused by foodborne pathogens are also significant global public health issues [ 3 , 4 ] . Salmonella is a gram-negative and facultatively anaerobic bacterium [ 5 , 6 ] , which can contaminate various food samples and survive for weeks or even years under favorable environmental conditions [ 7 ] . The reported contamination accidents of Salmonella are the highest among all pathogens. Salmonella is widely distributed and can infect a wide range of animals and human hosts [ 8 ] , causing human diseases including gastroenteritis [ 9 ] , bacteremia, and other extraintestinal infections [ 10 ] . The wide spread of Salmonella makes its control and prevention of great significance [ 11 – 13 ] . Rapid and accurate screening of Salmonella is crucial for effectively guaranteeing food safety. The traditional gold standard method for Salmonella detection is still the plate culture protocol [ 14 ] . The long conclusion-made time, tedious and multiple operation steps determine that this method is unsuitable for on-site screening of tremendous food samples [ 15 ] . Some methods have been developed for meeting the requirement of rapid detection, including mass spectrometry [ 16 , 17 ] , spectroscopy [ 18 ] , optical phenotyping [ 19 – 21 ] . The sensitivity of these methods for the whole bacteria detection is far from meeting the practical application requirements. The immune-recognition based methods provide both the efficiency and better sensitivity for applications. However, the current categories of antibody cannot meet the requirement for serotype identification of Salmonella [ 22 ] . Comparatively, the molecular methods could rapidly detect target Salmonella with specificity to different serotypes. However, the method’s efficiency, specificity and the convenience need further improvement for practical applications. For PCR related protocols, the nested PCR could significantly improve the efficiency and specificity of the amplification by the two primer sets based two-round amplification [ 23 , 24 ] . Traditional nested PCR is conducted by adding the amplicons of the first-round into the system of the second-round amplification [ 25 – 27 ] , which holds the potential risk of cross-contamination and the aerosol contamination. Besides, to improve the sensitivity and the specificity of the detection, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein (Cas) system has been extensively reported as the novel biological tool in gene editing and transcriptional regulation [ 28 , 29 ] . This system has been widely used for the detection of various foodborne pathogens including Listeria monocytogenes [ 30 ] , Escherichia coli [ 31 ] , Staphylococcus aureus [ 32 ] , and Salmonella [ 33 ] based on the trans -cleavage activity of the CRISPR/Cas system. For the final signal reporting, the lateral flow strip provides a simple and direct visual mode for rapid detection, which has been widely used in food safety [ 34 ] , and epidemic control [ 35 ] . Actually, the sensitivity and specificity of the lateral flow strip are also facing various challenges. The CRISPR/Cas has been integrated with lateral flow strip to improve the sensing performance including the sensitivity and specificity especially for inhibiting false-positive results [ 36 ] . Based on all of these, in this study, we have developed the one-tube nested PCR with the designed outer and inner primer sets to improve sensing performance. The CRISPR/Cas12a is further integrated with the one-tube nest PCR for ultrasensitive detection of Salmonella in the visible mode with fluorescent lateral flow strip, which has been successfully constructed for the rapid screening of Salmonella . 2. Materials and Methods 2.1 Materials and Reagents All bacterial strains used are sourced from the Anhui Provincial Product Quality Supervision and Inspection Research Institute. The primers used (detailed sequences, Table S1 ) were purchased from General Biosystems Co., Ltd. (Anhui, China). The bacterial genomic DNA extraction kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Cas12a protein was purchased from NEB. Absorbent pads, fiberglass membranes, nitrocellulose membranes (NC membranes), and PVC backing boards were purchased from Jie-Ning Biotech. Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA, 98%) was purchased from Biodee Inc. (Beijing, China). Fluorescein isothiocyanate antibody (FITC) was purchased from Abgree Biotechnology Co., Ltd. (Guangzhou, China). Sheep anti-mouse secondary antibody was purchased from Goodhere Biotechnology Co., Ltd. (Hangzhou, China). Casein was purchased from J&K Scientific Ltd. (Beijing, China). Sodium chloride (NaCl), sodium hydroxide (NaOH), concentrated hydrochloric acid (HCl), sucrose, polyethylene glycol-20000 (PEG-20000), and alginate were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Tween-20 and Triton X-100 were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Streptavidin (SAV), TE, agarose (hypotonic), 4S Red Plus nucleic acid dye, ddH 2 O, and PCR Mix were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). 2.2 Extraction of genomic DNA for analysis The bacterial solution was mixed thoroughly and 1 mL of bacterial solution was added to 9 mL sterile 0.85% NaCl. The above operation was repeated to dilute the bacterial solution in 10-fold concentration gradient. Bacterial genomic DNA extraction kit was utilized for DNA extraction according to the manufacturer's instructions. The DNA templates obtained were stored at -20°C for use. 2.3 Preparation of fluorescent microsphere (FM)-antibody conjugates of LFS To prepare the FM-antibody conjugate, 40 µL of diluted FM was sonicated for 5 min and added to 200 µL of 5 mM BB solution, which was further activated with 0.1 mg/mL EDC and 0.1 mg/mL NHS. The mixture was incubated at room temperature for 30 min. After centrifugation at 13,000 rpm for 15 min, the precipitate was re-dissolved in 200 µL of 5 mM BB and the appropriate amount of antibody was added and incubated at room temperature for 1 h. Then 0.5% Casein was added and reacted for another 1 h to block the unreacted sites of FM to avoid nonspecific adsorption. Finally, the precipitate was re-dissolved in 50 µL of resuspension solution by centrifugation at 13,000 rpm for 15 min. The obtained FM-antibody conjugates were sprayed onto the conjugation pad and dried at 37°C overnight for the assembly of LFS. 2.4 Design and assembly of lateral flow strip for rapid screening of Salmonella The sample pad was pretreated with the sample treatment solution (0.05 M Tris-HCl, 0.15 M NaCl, 0.25% Triton X-100). The conjugate pad for loading with FM-antibody conjugates was pretreated with solution (10 mM PB pH 7.4, 5% sucrose, 1% trehalose, 0.3% Tween-20, and 0.25% PEG 20000). The NC membrane was sprayed with control line (C) and test line (T) with goat anti-mouse secondary antibody line and SAV, respectively. The sample pads, coupling pads, nitrocellulose membranes, and absorbent pads were assembled onto the PVC backing card in sequence with the 2 mm overlap with each other. 2.5 Construction and optimization of one-tube nested PCR and CRISPR/Cas12a mediated LFS for rapid screening of Salmonella The one-tube nested PCR was conducted in a total volume of 25 µL including 12.5 µL of PCR mix, 1 µL of Salmonella DNA template, 10.5 µL of ultrapure deionized water, 200 nM of 188-F/188-R external primer set, and 200 nM of 64-F/64-R internal primer set. PCR was performed under the following conditions: denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 56.5°C for 30 s, and extension at 72°C for 30 s. The final extension was performed at 72°C for 5 min. Ultrapure deionized water without target template was adopted as the blank control for amplification. The amplicons of the nested PCR were added to the CRISPR/Cas12a system with the volume of 20 µL (10 nM reporter probe, 100 nM Cas protein, 200 nM crRNA and 2 µL Buffer r2.1). The mixture was incubated at 37°C for 50 min and followed by denaturation at 80°C for 10 min. Then, 2 µL of the reacted product and 48 µL of running buffer were dropped onto the sample pad of LFS for on-site visual measurement. 2.6 Specificity and sensitivity verification of the one-tube nested PCR and CRISPR/Cas12a assisted LFS DNA templates of Pseudomonas aeruginosa , Staphylococcus aureus , Vibrio parahaemolyticus , Escherichia coli , and Listeria monocytogenes were used as an interfering and control groups to assess the specificity of the one-tube nested PCR and CRISPR/Cas12a assisted LFS. The sensitivity of one-tube nested PCR and CRISPR/Cas12a assisted LFS was evaluated using the extracted Salmonella DNA at different concentrations (10 5 -10 0 CFU/mL). Nuclease-free water was used as template for the no template control (NTC). 2.7 Repeatability and stability verification Different batches of LFS were prepared under the same conditions for repeatability studies. The same batch of LFS was stored at room temperature and protected from light for 1, 7, 14, and 30 days. The above sets of LFS were validated using the extracted DNA of Salmonella at 10 2 CFU/mL, and the fluorescence signal intensity of the T-line was analyzed and compared using the ImageJ software. 2.8 Detection of Salmonella in practical food samples Milk and juice were purchased from the local supermarket as practical samples to validate the applicability of this method. In a sterile environment, 1 mL of Salmonella Typhimurium suspension at different concentrations (10 6 -10 0 CFU/mL) was added into 9 mL of samples with vortex. Then, the extract DNA template was measured with the one-tube nested PCR and CRISPR/Cas12a assisted LFS to evaluate the sensing performance of Salmonella Typhimurium in practical samples. 3. Results and Discussion 3.1 Sensing principal design of the one-tube nested PCR and CRISPR/Cas12a assisted LFS for ultrasensitive and accurate detection of Salmonella. As shown in Scheme 1 , the core design of this study focuses on integrating three critical aspects forimproving final sensing performance: (1) Design of one-tube nested PCR for efficiency improvement. The design of two primer sets including the external and internal primer sets makes the nested PCR well operated. In the one-tube mode, with the presence of the target Salmonella , the first-round amplicons of the external primer set could simultaneously act as the template for the internal primer set, which could greatly improve the amplification efficiency. (2) Both the amplicon sequence of the internal primer set and external primer set could act as the target of the CRISPR-Cas12a system to activate the trans -cleavage property, which will boost the cleavage of Cas. The sequence-dependent activation of cleavage also contributes to the liability of specificity. (3) All boosted cleavage products could be measured qualitatively and quantitatively with the fluorescent lateral flow strip in the visible mode. The FITC and biotin dual-labeled ssDNA probe will be randomly hydrolyzed into pieces by the trans -cleavage effect of activated Cas12a. For qualitative visual screening, only the C line shows the fluorescent signal on LFS as positive results while both the T line and C line will both show the fluorescent signal under the negative conditions. Besides, the quantitative analysis of target Salmonella can be realized based on the relationship between the concentration of and the fluorescent strength on T line of LFS. 3.2 Feasibility verification of the designed one-tube nested PCR and CRISPR/Cas12a mediated fluorescent LFS for Salmonella detection It is a common sense that the nested PCR highly depends on the design of external and internal primer sets. Based on the designed primer sets for Salmonella detection, simultaneous amplification results were first confirmed by the traditional 2% agarose gel electrophoresis (Fig. 1 A). Results in Fig. 1 A indicate that both the amplicons of 188 bp and 64 bp could be well amplified in the same reaction tube without any cross-reactivity, ensuring the accurate amplification of the target gene and providing an enormous template for activation of CRISPR/Cas system. More importantly, the efficiency of the nested results in Fig. 1 A is obviously higher than the traditional single PCR, demonstrating the improved amplification for further sensing as designed. As demonstrated in Scheme 1 , with the designed crRNA and the amplicons of the nested PCR, the CRIPSR/Cas12a system can be activated and the dual-labeled ssDNA reporter probe will be cleaved by the trans -cleavage effect. Results in Fig. 1 C clearly show that all the amplicons (external, internal, external & internal) could activate the trans -cleavage effect of CRISPR-Cas12a and induce the recovering of the fluorescent signals of the dual-labeled FAM-ssDNA-BHQ reporter probe (only for fluorescent recovery research). More interestingly, it is noted that the recovered fluorescent signal of the nested condition is the best ( ∆Rn3 ) and higher than the other two separated external ( ∆Rn2 ) and internal groups ( ∆Rn1 ), strongly indicating the improved sensing performance of the designed one-tube nest PCR. Finally, the final results are output and confirmed with the fluorescent LFS (Fig. 1 B). Due to the trans -cleaved of the dual-labeled FITC-ssDNA-biotin reporter probe, the fluorescent signal will be decreased accordingly. Among all three groups, in contrast, the external primer set group with the weaker variation on T line and the internal primer set group with induced a little higher fluorescent signal variation on the T line compared with negative groups. In comparison, the one-tube nest PCR with both the internal and external primer sets group had the strongest variation on the T line. All these results in Fig. 1 demonstrate that the designed one-tube nested PCR could contribute effectively to the sensing signal improvement for Salmonella detection. 3.3 Optimization of conditions for CRISPR/Cas12a cleavage system activation and fluorescent LFS screening. To achieve the optimal detection performance of this one-tube nested PCR and CRISPR/Cas co-mediated LFS for accurate and sensitive screening of Salmonella , various parameters of these two critical processes should be optimized. For the CRISPR/Cas system, the concentration of Cas12a and crRNA has been considered and 100 nM and 200 nM have been treated as the best for theactivating the cleavage effect. Besides, the cleavage time, temperature and the buffer category have also been investigated and 37°C, 50 min in the buffer r2.1 have been obtained for the best activation reaction. Meanwhile, the concentration of the dual-labeled FITC-ssDNA-biotin probe has been testified due to the fact that too high concentration will result in insufficient cleavage efficiency of the ssDNA probe and poor signal variation on the T line of LFS. On the contrary, with too low concentration of ssDNA probe, the signal on T line of the negative groups will also be too weak to produce sufficient signal for analysis. Finally, 10 nM has been treated as the optimal concentration for probe cleavage and signal reporting on the T line of LFS. The conditions for LFS measurements were also conducted for the improvement of sensing performance. The activation pH of surface groups on the fluorescent microspheres and the pH for the conjugation of antibodies onto the microspheres have been selected as 7.4 and 8.0, respectively. 0.5% casein was chosen as the blocking agent to avoid the nonspecific adsorption effect. All these optimized results could be found in detail in the supporting information ( Figure S2 ). 3.4 Evaluation of Salmonella sensing performance with the designed one-tube nested PCR and CRISPR/Cas12a co-mediated fluorescent LFS Target DNA extracted from the Salmonella samples at different concentrations (6×10 5 -6×10 0 CFU/mL) was determined with the one-tube nested PCR and CRISPR/Cas12a co-mediated fluorescent LFS. Results in Fig. 2 A demonstrate that, comparatively, the one-tube nested PCR could induce the visual distinguishable signal variation on the T line of the LFS at the concentration of 10 2 CFU/mL. In contrast,the traditional single PCR only induce the distinguishable signal variation at the concentration of 10 3 (internal primer set) and 10 4 (external primer set) CFU/mL, respectively. This means with the design and application of one-tube nested PCR, the visual detection limit is improved at least 10 and 100 times compared with traditional single PCR. For the quantitative analysis, the detection limit of the one-tube nested PCR and CRISPR/Cas12a co-mediated LFS is as low as 10 1 CFU/mL, while the traditional single PCR of the 10 2 (internal primer set) and 10 3 (external primer set) CFU/mL, respectively. Besides, the linear sensing curve was also constructed with the satisfied linear relationship between the concentration of the bacteria and the fluorescent signal on the T line of LFS (Fig. 2 B) in the range of 6×10 5 -6×10 0 CFU/mL with R 2 of 0.981 (Fig. 2 C). As one of the valuable properties of the new method, the specificity of this one-tube nested PCR and CRISPR/Cas12a assisted LFS was verified with other common pathogens, including the Pseudomonas aeruginosa , Staphylococcus aureus , Vibrio parahaemolyticus , and Escherichia coli , Listeria monocytogenes . Results in Fig. 3 show that only the presence of target Salmonella can induce the obvious inhibition of the fluorescent signal on T line of LFS, while other control pathogens cannot. Even the mixture of all the above-mentioned control pathogens cannot induce any signal decays on T line of LFS, indicating the excellent specificity of the designed method. This excellent specificity can be ascribed to the designed nested PCR and the amplicon-activated trans -cleavage of CRISPR/Cas. This designed one-tube nested PCR and CRISPR/Cas12a co-mediated LFS was batch-prepared and stored for different times to verify the repeatability and stability of the method. Firstly, ten LFS of the same batch were adopted to detect the same Salmonella sample at the concentration of 6×10 2 CFU/mL. As shown in Fig. 4 , the fluorescent signal on T line was analyzed and compared. The ten parallel measurements relative standard deviation (RSD) is 1.64%, indicating the satisfactory practical applications repeatability. The prepared LFS were stored for 1, 7, 14 and 30 days and then used to detect the same sample, and the fluorescent signals on the T line were compared as shown in Fig. 4 B. There are no obvious signal deviations of LFS with different stored time, and the coefficient variation is calculated as 5.98%, demonstrating the acceptable stability of the method for practical measurements. 3.5 Practical detection of target Salmonella in food samples with this one-tube nested PCR and CRISPR/Cas12a mediated LFS Finally, the practical food samples were verified with this designed one-tube nested PCR and CRISP/cas12a co-mediated LFS. Salmonella Typhimurium at the concentration of 6×10 3 CFU/mL was spiked into the pre-confirmed negative milk and juice samples and measured with this new method. Results in Fig. 5 clearly show that both spiked milk and juice samples are measured as positive with the obvious signal inhibition on the T line of LFS. The recovery studies were also performed, as demonstrated in Table 1 . Different concentrations of target Salmonella were spiked into the different food samples with different matrix effects. The results in Table 1 indicate that the recoveries are achieved in the range of 98.24–118.57% with the RSD of 1.65–6.89%. All these qualitative and quantitative application results greatly support the practical application of this one-tube nested PCR and CRISPR/Cas12a co-mediated LFS in real food samples. Table 1 Recoveries of LFA for Salmonella detection in milk and juice (n = 3). Sample Spiked (CFU/mL) RSD (%) Recovery (%) milk 10 2 6.89 98.24 10 3 4.75 112.71 juice 10 2 2.23 111.60 10 3 1.65 118.57 4. Conclusion In this study, a one-tube nested PCR and CRISPR/Cas12a co-mediated fluorescent LFS was designed and developed for sensitive and accurate detection of Salmonella in the visible mode. With the well designed and optimized external and internal primer sets, the one-tube nested PCR was constructed to amplify the target gene of Salmonella , which greatly improved the amplification efficiency without any cross-interference. All produced amplicons further activated the trans -cleavage property of CRISPR/Cas12a for signal output, which greatly ensured the specificity of the whole detection. Finally, the output signals were recorded with the fluorescent LFS in the visible mode. With the integration of these designed strategies, as low as 10 1 CFU/mL could be well determined in food samples. The practical food samples have also been well measured, and satisfactory results have been achieved. This one-tube nested PCR greatly improved the efficiency of amplification and the detection sensitivity, while the CRIPSR/Cas12a effectively guaranteed the specificity. The fluorescent LFS showed the qualitative and quantitative results in the simple visible mode. This one-tube nested PCR and CRISPR/Cas12a co-mediated LFS holds great promise in the rapid and accurate detection of Salmonella and can be extended to other common pathogens with new primer set and crRNA design in food safety and clinical diagnosis. Declarations Funding This work was supported by the grants received from the National Key Research Program (2024YFF0618101), the NSFC program (32172295), and the Hainan Key Research Program (ZDYF2022XDNY248). Data availability Not applicable. Ethical approval Not applicable. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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(2022) Fluorescent microspheres lateral flow assay integrated with Smartphone-based reader for multiple microRNAs detection. Microchemical Journal 179: 107551. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx originalimageofgelelectrophoresis.docx graphicabstract.jpg Scheme1.png Scheme 1. The schematic diagram of rapid identification of Salmonella with the one-tube nested PCR and CRISPR-Cas12 assisted LFS. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7534316","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515279555,"identity":"83284b24-b553-4043-bb52-937e94bb63f9","order_by":0,"name":"Qingli Yang","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Qingli","middleName":"","lastName":"Yang","suffix":""},{"id":515279556,"identity":"b798521f-dc37-455d-a3e5-339a063eeb3e","order_by":1,"name":"Noor Fatima","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Noor","middleName":"","lastName":"Fatima","suffix":""},{"id":515279557,"identity":"f4497e67-12cc-4c85-b702-3aaf7c28b869","order_by":2,"name":"Xiaofeng Yu","email":"","orcid":"","institution":"Anhui Province Institute of Product Quality Supervision \u0026 Inspection","correspondingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Yu","suffix":""},{"id":515279563,"identity":"7445c9dc-52f7-4ecc-bc45-d7fa7cdfdf6a","order_by":3,"name":"Yubo Peng","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yubo","middleName":"","lastName":"Peng","suffix":""},{"id":515279567,"identity":"888dcb7f-66ec-44dd-831d-f5a732c29094","order_by":4,"name":"Qi Chen","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Chen","suffix":""},{"id":515279568,"identity":"e57e3ce7-76e8-418b-941f-17f3c7eb2583","order_by":5,"name":"Dexing Zeng","email":"","orcid":"","institution":"Anhui Province Institute of Product Quality Supervision \u0026 Inspection","correspondingAuthor":false,"prefix":"","firstName":"Dexing","middleName":"","lastName":"Zeng","suffix":""},{"id":515279569,"identity":"c6ac08bc-0058-4671-8a05-721a89449f35","order_by":6,"name":"Zhaoran Chen","email":"","orcid":"","institution":"Anhui Province Institute of Product Quality Supervision \u0026 Inspection","correspondingAuthor":false,"prefix":"","firstName":"Zhaoran","middleName":"","lastName":"Chen","suffix":""},{"id":515279570,"identity":"cc845f08-0d52-48fa-9f8f-98aadb51b557","order_by":7,"name":"Guolin Wu","email":"","orcid":"","institution":"University of Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Guolin","middleName":"","lastName":"Wu","suffix":""},{"id":515279571,"identity":"c1543b73-f51e-43d7-8f60-07bb117f18ed","order_by":8,"name":"Wei Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYDACCShpwMDA+CChooY0LcwGD84cI1oLA0gLm+TDFmbCOvhnNx97zFNhYW8u3WNWkdjAxsDf3p2A35I7x9KNec5IJO6cc8bsRuIOGQaJM2c34NViIJFjJp3bJpFgcCMHqOUMG1Akl5CW/G/Suf8k7EFaChLbmInRksMmndsgwbgBqIWBKC0SN9LMpP8ck0jccCOtWCLhzDEegn7hn5H8THJGTR3QYckbP/6oqJHjb+/FrwUD8JCmfBSMglEwCkYBVgAARaVE9GZcuoEAAAAASUVORK5CYII=","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Wei","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-09-04 09:08:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7534316/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7534316/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91663342,"identity":"465bcd1c-0cc0-4843-871e-c4f894164a2a","added_by":"auto","created_at":"2025-09-18 22:58:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1394394,"visible":true,"origin":"","legend":"\u003cp\u003eFeasibility verification results. (A) Agarose gel electrophoresis (AGE) of PCR amplicons with a single primer pair and two primer pairs. 1-2 lanes: Inner primers (1: blank, 2: \u003cem\u003eSalmonella\u003c/em\u003e template), 3-4 lanes: Outer primers (3: blank, 4: \u003cem\u003eSalmonella\u003c/em\u003etemplate), 5-6 lanes: Inner primers and Outer primers (5: blank, 6: \u003cem\u003eSalmonella\u003c/em\u003etemplate). Single-primer amplicons and double-primer amplicons activate the LFA signal (B) and fluorescence signal (C) generated by the CRISPR-Cas12a system. ∆Rn: Difference in fluorescence intensity between negative and positive (∆Rn1: Inner primers, ∆Rn2: Outer primers, ∆Rn3: Inner primers and Outer primers).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7534316/v1/8d058842a7bc7133a9183b89.png"},{"id":91663134,"identity":"dff93184-b6a1-4e52-b6cd-c777c8410c38","added_by":"auto","created_at":"2025-09-18 22:50:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":190887,"visible":true,"origin":"","legend":"\u003cp\u003eSensitivity of the one-tube nested PCR-CRISPR/Cas12a-LFS-based assay. (A) The results of test strips for detecting different concentrations of Salmonella typhimurium samples. From left to right: 6×10\u003csup\u003e5\u003c/sup\u003e, 6×10\u003csup\u003e4\u003c/sup\u003e, 6×10\u003csup\u003e3\u003c/sup\u003e, 6×10\u003csup\u003e2\u003c/sup\u003e, 6×10\u003csup\u003e1\u003c/sup\u003e, 6×10\u003csup\u003e0\u003c/sup\u003e, 0 CFU/mL. (B) Fluorescence intensity of C and T lines at different concentrations. (C) Linear analysis results of the dual-primer assay (R\u003csup\u003e2\u003c/sup\u003e=0.981). (a) Linear analysis results of the Inner primer assay (R\u003csup\u003e2\u003c/sup\u003e=0.978). (b) Linear analysis results of the External primer assay (R\u003csup\u003e2\u003c/sup\u003e=0.965).\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7534316/v1/7ce07b5ae5e79cedf35e6c9f.png"},{"id":91663136,"identity":"96cb743a-9224-4634-b57f-24bdd507b7c5","added_by":"auto","created_at":"2025-09-18 22:50:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":564662,"visible":true,"origin":"","legend":"\u003cp\u003eSpecificity testing of one-tube nested PCR-CRISPR/Cas12a-LFS for \u003cem\u003eSalmonella\u003c/em\u003e targets. From left to right: \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e, and \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eListeria monocytogenes\u003c/em\u003e,\u003cem\u003e \u003c/em\u003emixed bacteria except\u003cem\u003e Salmonella.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7534316/v1/cfc2134b338f30d7104c2101.png"},{"id":91663341,"identity":"a185a94f-0bfd-42f1-99d6-34290c67c74b","added_by":"auto","created_at":"2025-09-18 22:58:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":376616,"visible":true,"origin":"","legend":"\u003cp\u003eRepeatability and stability verification. (A) Results of test strips from 10 replicate experiments; (B) T-line intensities of test strips stored for one month at room temperature and protected from light.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7534316/v1/ac658093db5de15c4211b148.png"},{"id":91663345,"identity":"c72ad529-9b91-44c0-9e74-91c8f08e19fc","added_by":"auto","created_at":"2025-09-18 22:58:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":321990,"visible":true,"origin":"","legend":"\u003cp\u003eDetection results of \u003cem\u003eSalmonella\u003c/em\u003ein milk and juice samples.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7534316/v1/47566e8644a936f7ed22541a.png"},{"id":103975243,"identity":"2ec9becc-95a9-4d30-8c3a-8a1bca6e09c7","added_by":"auto","created_at":"2026-03-05 08:27:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3894251,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7534316/v1/ea569714-fafc-42ba-bb62-9c7b5fac6862.pdf"},{"id":91663344,"identity":"134058f7-1d27-4d8d-9ee8-fecda9cee81e","added_by":"auto","created_at":"2025-09-18 22:58:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":306578,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7534316/v1/a8dd888984b6472c0600b1d9.docx"},{"id":91663141,"identity":"38304ed1-e1b3-4444-a133-bba227e2da04","added_by":"auto","created_at":"2025-09-18 22:50:36","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1323588,"visible":true,"origin":"","legend":"","description":"","filename":"originalimageofgelelectrophoresis.docx","url":"https://assets-eu.researchsquare.com/files/rs-7534316/v1/1d269aa158fd4deb0db7883b.docx"},{"id":91663137,"identity":"17c1a884-5bc7-4fd2-b26c-76e8bff246ff","added_by":"auto","created_at":"2025-09-18 22:50:36","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":66615,"visible":true,"origin":"","legend":"","description":"","filename":"graphicabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7534316/v1/6162aceb47fbe3848b1c7da4.jpg"},{"id":91663427,"identity":"1caa87d5-6092-4186-9063-c93b7b9a9e17","added_by":"auto","created_at":"2025-09-18 23:06:36","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":215210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eThe schematic diagram of rapid identification of \u003cem\u003eSalmonella\u003c/em\u003e with the one-tube nested PCR and CRISPR-Cas12 assisted LFS.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-7534316/v1/7944aee7fa0c6f2ace97b49e.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"One-tube nested PCR mediated CRISPR-Cas12a: A novel performance-enhanced approach for ultrasensitive fluorescent visual screening of Salmonella","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMicrobial contamination could occur during the growth, harvest, preparation, storage, processing, and distribution of food\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Foodborne pathogens can cause illness or even death of consumers\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Diseases caused by foodborne pathogens are also significant global public health issues\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eSalmonella\u003c/em\u003e is a gram-negative and facultatively anaerobic bacterium\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, which can contaminate various food samples and survive for weeks or even years under favorable environmental conditions\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The reported contamination accidents of \u003cem\u003eSalmonella\u003c/em\u003e are the highest among all pathogens. \u003cem\u003eSalmonella\u003c/em\u003e is widely distributed and can infect a wide range of animals and human hosts\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, causing human diseases including gastroenteritis\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, bacteremia, and other extraintestinal infections\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The wide spread of \u003cem\u003eSalmonella\u003c/em\u003e makes its control and prevention of great significance \u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Rapid and accurate screening of \u003cem\u003eSalmonella\u003c/em\u003e is crucial for effectively guaranteeing food safety.\u003c/p\u003e\u003cp\u003eThe traditional gold standard method for \u003cem\u003eSalmonella\u003c/em\u003e detection is still the plate culture protocol \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The long conclusion-made time, tedious and multiple operation steps determine that this method is unsuitable for on-site screening of tremendous food samples\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Some methods have been developed for meeting the requirement of rapid detection, including mass spectrometry\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, spectroscopy\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, optical phenotyping\u003csup\u003e[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. The sensitivity of these methods for the whole bacteria detection is far from meeting the practical application requirements. The immune-recognition based methods provide both the efficiency and better sensitivity for applications. However, the current categories of antibody cannot meet the requirement for serotype identification of \u003cem\u003eSalmonella\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Comparatively, the molecular methods could rapidly detect target \u003cem\u003eSalmonella\u003c/em\u003e with specificity to different serotypes. However, the method\u0026rsquo;s efficiency, specificity and the convenience need further improvement for practical applications.\u003c/p\u003e\u003cp\u003eFor PCR related protocols, the nested PCR could significantly improve the efficiency and specificity of the amplification by the two primer sets based two-round amplification\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Traditional nested PCR is conducted by adding the amplicons of the first-round into the system of the second-round amplification\u003csup\u003e[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, which holds the potential risk of cross-contamination and the aerosol contamination. Besides, to improve the sensitivity and the specificity of the detection, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein (Cas) system has been extensively reported as the novel biological tool in gene editing and transcriptional regulation\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. This system has been widely used for the detection of various foodborne pathogens including \u003cem\u003eListeria monocytogenes\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, and \u003cem\u003eSalmonella\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e based on the \u003cem\u003etrans\u003c/em\u003e-cleavage activity of the CRISPR/Cas system. For the final signal reporting, the lateral flow strip provides a simple and direct visual mode for rapid detection, which has been widely used in food safety\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, and epidemic control\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Actually, the sensitivity and specificity of the lateral flow strip are also facing various challenges. The CRISPR/Cas has been integrated with lateral flow strip to improve the sensing performance including the sensitivity and specificity especially for inhibiting false-positive results\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Based on all of these, in this study, we have developed the one-tube nested PCR with the designed outer and inner primer sets to improve sensing performance. The CRISPR/Cas12a is further integrated with the one-tube nest PCR for ultrasensitive detection of \u003cem\u003eSalmonella\u003c/em\u003e in the visible mode with fluorescent lateral flow strip, which has been successfully constructed for the rapid screening of \u003cem\u003eSalmonella\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials and Reagents\u003c/h2\u003e\u003cp\u003eAll bacterial strains used are sourced from the Anhui Provincial Product Quality Supervision and Inspection Research Institute. The primers used (detailed sequences, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) were purchased from General Biosystems Co., Ltd. (Anhui, China). The bacterial genomic DNA extraction kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Cas12a protein was purchased from NEB. Absorbent pads, fiberglass membranes, nitrocellulose membranes (NC membranes), and PVC backing boards were purchased from Jie-Ning Biotech. Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA, 98%) was purchased from Biodee Inc. (Beijing, China). Fluorescein isothiocyanate antibody (FITC) was purchased from Abgree Biotechnology Co., Ltd. (Guangzhou, China). Sheep anti-mouse secondary antibody was purchased from Goodhere Biotechnology Co., Ltd. (Hangzhou, China). Casein was purchased from J\u0026amp;K Scientific Ltd. (Beijing, China). Sodium chloride (NaCl), sodium hydroxide (NaOH), concentrated hydrochloric acid (HCl), sucrose, polyethylene glycol-20000 (PEG-20000), and alginate were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Tween-20 and Triton X-100 were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Streptavidin (SAV), TE, agarose (hypotonic), 4S Red Plus nucleic acid dye, ddH\u003csub\u003e2\u003c/sub\u003eO, and PCR Mix were purchased from Sangon Biotech Co., Ltd. (Shanghai, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Extraction of genomic DNA for analysis\u003c/h2\u003e\u003cp\u003eThe bacterial solution was mixed thoroughly and 1 mL of bacterial solution was added to 9 mL sterile 0.85% NaCl. The above operation was repeated to dilute the bacterial solution in 10-fold concentration gradient. Bacterial genomic DNA extraction kit was utilized for DNA extraction according to the manufacturer's instructions. The DNA templates obtained were stored at -20\u0026deg;C for use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Preparation of fluorescent microsphere (FM)-antibody conjugates of LFS\u003c/h2\u003e\u003cp\u003eTo prepare the FM-antibody conjugate, 40 \u0026micro;L of diluted FM was sonicated for 5 min and added to 200 \u0026micro;L of 5 mM BB solution, which was further activated with 0.1 mg/mL EDC and 0.1 mg/mL NHS. The mixture was incubated at room temperature for 30 min. After centrifugation at 13,000 rpm for 15 min, the precipitate was re-dissolved in 200 \u0026micro;L of 5 mM BB and the appropriate amount of antibody was added and incubated at room temperature for 1 h. Then 0.5% Casein was added and reacted for another 1 h to block the unreacted sites of FM to avoid nonspecific adsorption. Finally, the precipitate was re-dissolved in 50 \u0026micro;L of resuspension solution by centrifugation at 13,000 rpm for 15 min. The obtained FM-antibody conjugates were sprayed onto the conjugation pad and dried at 37\u0026deg;C overnight for the assembly of LFS.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Design and assembly of lateral flow strip for rapid screening of \u003cem\u003eSalmonella\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe sample pad was pretreated with the sample treatment solution (0.05 M Tris-HCl, 0.15 M NaCl, 0.25% Triton X-100). The conjugate pad for loading with FM-antibody conjugates was pretreated with solution (10 mM PB pH 7.4, 5% sucrose, 1% trehalose, 0.3% Tween-20, and 0.25% PEG 20000). The NC membrane was sprayed with control line (C) and test line (T) with goat anti-mouse secondary antibody line and SAV, respectively. The sample pads, coupling pads, nitrocellulose membranes, and absorbent pads were assembled onto the PVC backing card in sequence with the 2 mm overlap with each other.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.5 Construction and optimization of one-tube nested PCR and CRISPR/Cas12a mediated LFS for rapid screening of\u003c/b\u003e \u003cb\u003eSalmonella\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe one-tube nested PCR was conducted in a total volume of 25 \u0026micro;L including 12.5 \u0026micro;L of PCR mix, 1 \u0026micro;L of \u003cem\u003eSalmonella\u003c/em\u003e DNA template, 10.5 \u0026micro;L of ultrapure deionized water, 200 nM of 188-F/188-R external primer set, and 200 nM of 64-F/64-R internal primer set. PCR was performed under the following conditions: denaturation at 95\u0026deg;C for 5 min, followed by 30 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 56.5\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 30 s. The final extension was performed at 72\u0026deg;C for 5 min. Ultrapure deionized water without target template was adopted as the blank control for amplification.\u003c/p\u003e\u003cp\u003eThe amplicons of the nested PCR were added to the CRISPR/Cas12a system with the volume of 20 \u0026micro;L (10 nM reporter probe, 100 nM Cas protein, 200 nM crRNA and 2 \u0026micro;L Buffer r2.1). The mixture was incubated at 37\u0026deg;C for 50 min and followed by denaturation at 80\u0026deg;C for 10 min. Then, 2 \u0026micro;L of the reacted product and 48 \u0026micro;L of running buffer were dropped onto the sample pad of LFS for on-site visual measurement.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Specificity and sensitivity verification of the one-tube nested PCR and CRISPR/Cas12a assisted LFS\u003c/h2\u003e\u003cp\u003eDNA templates of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eListeria monocytogenes\u003c/em\u003e were used as an interfering and control groups to assess the specificity of the one-tube nested PCR and CRISPR/Cas12a assisted LFS. The sensitivity of one-tube nested PCR and CRISPR/Cas12a assisted LFS was evaluated using the extracted \u003cem\u003eSalmonella\u003c/em\u003e DNA at different concentrations (10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e0\u003c/sup\u003e CFU/mL). Nuclease-free water was used as template for the no template control (NTC).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Repeatability and stability verification\u003c/h2\u003e\u003cp\u003eDifferent batches of LFS were prepared under the same conditions for repeatability studies. The same batch of LFS was stored at room temperature and protected from light for 1, 7, 14, and 30 days. The above sets of LFS were validated using the extracted DNA of \u003cem\u003eSalmonella\u003c/em\u003e at 10\u003csup\u003e2\u003c/sup\u003e CFU/mL, and the fluorescence signal intensity of the T-line was analyzed and compared using the ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Detection of \u003cem\u003eSalmonella\u003c/em\u003e in practical food samples\u003c/h2\u003e\u003cp\u003eMilk and juice were purchased from the local supermarket as practical samples to validate the applicability of this method. In a sterile environment, 1 mL of \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e suspension at different concentrations (10\u003csup\u003e6\u003c/sup\u003e-10\u003csup\u003e0\u003c/sup\u003e CFU/mL) was added into 9 mL of samples with vortex. Then, the extract DNA template was measured with the one-tube nested PCR and CRISPR/Cas12a assisted LFS to evaluate the sensing performance of \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e in practical samples.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e3.1 Sensing principal design of the one-tube nested PCR and CRISPR/Cas12a assisted LFS for ultrasensitive and accurate detection of\u003c/strong\u003e \u003cstrong\u003eSalmonella.\u003c/strong\u003e\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eAs shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the core design of this study focuses on integrating three critical aspects forimproving final sensing performance: (1) Design of one-tube nested PCR for efficiency improvement. The design of two primer sets including the external and internal primer sets makes the nested PCR well operated. In the one-tube mode, with the presence of the target \u003cem\u003eSalmonella\u003c/em\u003e, the first-round amplicons of the external primer set could simultaneously act as the template for the internal primer set, which could greatly improve the amplification efficiency. (2) Both the amplicon sequence of the internal primer set and external primer set could act as the target of the CRISPR-Cas12a system to activate the \u003cem\u003etrans\u003c/em\u003e-cleavage property, which will boost the cleavage of Cas. The sequence-dependent activation of cleavage also contributes to the liability of specificity. (3) All boosted cleavage products could be measured qualitatively and quantitatively with the fluorescent lateral flow strip in the visible mode. The FITC and biotin dual-labeled ssDNA probe will be randomly hydrolyzed into pieces by the \u003cem\u003etrans\u003c/em\u003e-cleavage effect of activated Cas12a. For qualitative visual screening, only the C line shows the fluorescent signal on LFS as positive results while both the T line and C line will both show the fluorescent signal under the negative conditions. Besides, the quantitative analysis of target \u003cem\u003eSalmonella\u003c/em\u003e can be realized based on the relationship between the concentration of and the fluorescent strength on T line of LFS.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2 Feasibility verification of the designed one-tube nested PCR and CRISPR/Cas12a mediated fluorescent LFS for\u003c/b\u003e \u003cb\u003eSalmonella\u003c/b\u003e \u003cb\u003edetection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIt is a common sense that the nested PCR highly depends on the design of external and internal primer sets. Based on the designed primer sets for \u003cem\u003eSalmonella\u003c/em\u003e detection, simultaneous amplification results were first confirmed by the traditional 2% agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Results in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA indicate that both the amplicons of 188 bp and 64 bp could be well amplified in the same reaction tube without any cross-reactivity, ensuring the accurate amplification of the target gene and providing an enormous template for activation of CRISPR/Cas system. More importantly, the efficiency of the nested results in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA is obviously higher than the traditional single PCR, demonstrating the improved amplification for further sensing as designed.\u003c/p\u003e\u003cp\u003eAs demonstrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, with the designed crRNA and the amplicons of the nested PCR, the CRIPSR/Cas12a system can be activated and the dual-labeled ssDNA reporter probe will be cleaved by the \u003cem\u003etrans\u003c/em\u003e-cleavage effect. Results in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC clearly show that all the amplicons (external, internal, external \u0026amp; internal) could activate the \u003cem\u003etrans\u003c/em\u003e-cleavage effect of CRISPR-Cas12a and induce the recovering of the fluorescent signals of the dual-labeled FAM-ssDNA-BHQ reporter probe (only for fluorescent recovery research). More interestingly, it is noted that the recovered fluorescent signal of the nested condition is the best (\u003cb\u003e∆Rn3\u003c/b\u003e) and higher than the other two separated external (\u003cb\u003e∆Rn2\u003c/b\u003e) and internal groups (\u003cb\u003e∆Rn1\u003c/b\u003e), strongly indicating the improved sensing performance of the designed one-tube nest PCR.\u003c/p\u003e\u003cp\u003eFinally, the final results are output and confirmed with the fluorescent LFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Due to the \u003cem\u003etrans\u003c/em\u003e-cleaved of the dual-labeled FITC-ssDNA-biotin reporter probe, the fluorescent signal will be decreased accordingly. Among all three groups, in contrast, the external primer set group with the weaker variation on T line and the internal primer set group with induced a little higher fluorescent signal variation on the T line compared with negative groups. In comparison, the one-tube nest PCR with both the internal and external primer sets group had the strongest variation on the T line. All these results in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e demonstrate that the designed one-tube nested PCR could contribute effectively to the sensing signal improvement for \u003cem\u003eSalmonella\u003c/em\u003e detection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Optimization of conditions for CRISPR/Cas12a cleavage system activation and fluorescent LFS screening.\u003c/h2\u003e\u003cp\u003eTo achieve the optimal detection performance of this one-tube nested PCR and CRISPR/Cas co-mediated LFS for accurate and sensitive screening of \u003cem\u003eSalmonella\u003c/em\u003e, various parameters of these two critical processes should be optimized. For the CRISPR/Cas system, the concentration of Cas12a and crRNA has been considered and 100 nM and 200 nM have been treated as the best for theactivating the cleavage effect. Besides, the cleavage time, temperature and the buffer category have also been investigated and 37\u0026deg;C, 50 min in the buffer r2.1 have been obtained for the best activation reaction. Meanwhile, the concentration of the dual-labeled FITC-ssDNA-biotin probe has been testified due to the fact that too high concentration will result in insufficient cleavage efficiency of the ssDNA probe and poor signal variation on the T line of LFS. On the contrary, with too low concentration of ssDNA probe, the signal on T line of the negative groups will also be too weak to produce sufficient signal for analysis. Finally, 10 nM has been treated as the optimal concentration for probe cleavage and signal reporting on the T line of LFS.\u003c/p\u003e\u003cp\u003eThe conditions for LFS measurements were also conducted for the improvement of sensing performance. The activation pH of surface groups on the fluorescent microspheres and the pH for the conjugation of antibodies onto the microspheres have been selected as 7.4 and 8.0, respectively. 0.5% casein was chosen as the blocking agent to avoid the nonspecific adsorption effect. All these optimized results could be found in detail in the supporting information (\u003cb\u003eFigure S2\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.4 Evaluation of\u003c/b\u003e \u003cb\u003eSalmonella\u003c/b\u003e \u003cb\u003esensing performance with the designed one-tube nested PCR and CRISPR/Cas12a co-mediated fluorescent LFS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTarget DNA extracted from the \u003cem\u003eSalmonella\u003c/em\u003e samples at different concentrations (6\u0026times;10\u003csup\u003e5\u003c/sup\u003e-6\u0026times;10\u003csup\u003e0\u003c/sup\u003e CFU/mL) was determined with the one-tube nested PCR and CRISPR/Cas12a co-mediated fluorescent LFS. \u003cb\u003eResults in\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA demonstrate that, comparatively, the one-tube nested PCR could induce the visual distinguishable signal variation on the T line of the LFS at the concentration of 10\u003csup\u003e2\u003c/sup\u003e CFU/mL. In contrast,the traditional single PCR only induce the distinguishable signal variation at the concentration of 10\u003csup\u003e3\u003c/sup\u003e (internal primer set) and 10\u003csup\u003e4\u003c/sup\u003e (external primer set) CFU/mL, respectively. This means with the design and application of one-tube nested PCR, the visual detection limit is improved at least 10 and 100 times compared with traditional single PCR. For the quantitative analysis, the detection limit of the one-tube nested PCR and CRISPR/Cas12a co-mediated LFS is as low as 10\u003csup\u003e1\u003c/sup\u003e CFU/mL, while the traditional single PCR of the 10\u003csup\u003e2\u003c/sup\u003e (internal primer set) and 10\u003csup\u003e3\u003c/sup\u003e (external primer set) CFU/mL, respectively. Besides, the linear sensing curve was also constructed with the satisfied linear relationship between the concentration of the bacteria and the fluorescent signal on the T line of LFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) in the range of 6\u0026times;10\u003csup\u003e5\u003c/sup\u003e-6\u0026times;10\u003csup\u003e0\u003c/sup\u003e CFU/mL with R\u003csup\u003e2\u003c/sup\u003e of 0.981 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs one of the valuable properties of the new method, the specificity of this one-tube nested PCR and CRISPR/Cas12a assisted LFS was verified with other common pathogens, including the \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e, and \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eListeria monocytogenes\u003c/em\u003e. Results in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e show that only the presence of target \u003cem\u003eSalmonella\u003c/em\u003e can induce the obvious inhibition of the fluorescent signal on T line of LFS, while other control pathogens cannot. Even the mixture of all the above-mentioned control pathogens cannot induce any signal decays on T line of LFS, indicating the excellent specificity of the designed method. This excellent specificity can be ascribed to the designed nested PCR and the amplicon-activated \u003cem\u003etrans\u003c/em\u003e-cleavage of CRISPR/Cas.\u003c/p\u003e\u003cp\u003eThis designed one-tube nested PCR and CRISPR/Cas12a co-mediated LFS was batch-prepared and stored for different times to verify the repeatability and stability of the method. Firstly, ten LFS of the same batch were adopted to detect the same \u003cem\u003eSalmonella\u003c/em\u003e sample at the concentration of 6\u0026times;10\u003csup\u003e2\u003c/sup\u003e CFU/mL. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the fluorescent signal on T line was analyzed and compared. The ten parallel measurements relative standard deviation (RSD) is 1.64%, indicating the satisfactory practical applications repeatability. The prepared LFS were stored for 1, 7, 14 and 30 days and then used to detect the same sample, and the fluorescent signals on the T line were compared as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. There are no obvious signal deviations of LFS with different stored time, and the coefficient variation is calculated as 5.98%, demonstrating the acceptable stability of the method for practical measurements.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5 Practical detection of target\u003c/b\u003e \u003cb\u003eSalmonella\u003c/b\u003e \u003cb\u003ein food samples with this one-tube nested PCR and CRISPR/Cas12a mediated LFS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFinally, the practical food samples were verified with this designed one-tube nested PCR and CRISP/cas12a co-mediated LFS. \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e at the concentration of 6\u0026times;10\u003csup\u003e3\u003c/sup\u003e CFU/mL was spiked into the pre-confirmed negative milk and juice samples and measured with this new method. Results in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e clearly show that both spiked milk and juice samples are measured as positive with the obvious signal inhibition on the T line of LFS. The recovery studies were also performed, as demonstrated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Different concentrations of target \u003cem\u003eSalmonella\u003c/em\u003e were spiked into the different food samples with different matrix effects. The results in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicate that the recoveries are achieved in the range of 98.24\u0026ndash;118.57% with the RSD of 1.65\u0026ndash;6.89%. All these qualitative and quantitative application results greatly support the practical application of this one-tube nested PCR and CRISPR/Cas12a co-mediated LFS in real food samples.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRecoveries of LFA for \u003cem\u003eSalmonella\u003c/em\u003e detection in milk and juice (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpiked (CFU/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRSD (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRecovery (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003emilk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e98.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e112.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ejuice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e111.60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e118.57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, a one-tube nested PCR and CRISPR/Cas12a co-mediated fluorescent LFS was designed and developed for sensitive and accurate detection of \u003cem\u003eSalmonella\u003c/em\u003e in the visible mode. With the well designed and optimized external and internal primer sets, the one-tube nested PCR was constructed to amplify the target gene of \u003cem\u003eSalmonella\u003c/em\u003e, which greatly improved the amplification efficiency without any cross-interference. All produced amplicons further activated the \u003cem\u003etrans\u003c/em\u003e-cleavage property of CRISPR/Cas12a for signal output, which greatly ensured the specificity of the whole detection. Finally, the output signals were recorded with the fluorescent LFS in the visible mode. With the integration of these designed strategies, as low as 10\u003csup\u003e1\u003c/sup\u003e CFU/mL could be well determined in food samples. The practical food samples have also been well measured, and satisfactory results have been achieved. This one-tube nested PCR greatly improved the efficiency of amplification and the detection sensitivity, while the CRIPSR/Cas12a effectively guaranteed the specificity. The fluorescent LFS showed the qualitative and quantitative results in the simple visible mode. This one-tube nested PCR and CRISPR/Cas12a co-mediated LFS holds great promise in the rapid and accurate detection of \u003cem\u003eSalmonella\u003c/em\u003e and can be extended to other common pathogens with new primer set and crRNA design in food safety and clinical diagnosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the grants received from the National Key Research Program (2024YFF0618101), the NSFC program (32172295), and the Hainan Key Research Program (ZDYF2022XDNY248).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShao L, Sun Y, Zou B, et al. 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Microchemical Journal 179: 107551.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Salmonella, one-tube nested PCR, simultaneous amplification, signal enhancement, CRISPR/Cas, lateral flow strip, visual screening","lastPublishedDoi":"10.21203/rs.3.rs-7534316/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7534316/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e is one of the most prevalent and highly transmissible food-borne pathogens, making rapid and accurate screening essential for safeguarding human health and ensuring food safety. This study introduces a one-tube nested PCR mediated CRISPR-Cas12a for ultrasensitive visual screening of \u003cem\u003eSalmonella spp.\u003c/em\u003e using fluorescent lateral flow strip. By leveraging the simultaneous dual-segment amplification capability of the designed one-tube nested PCR and the collateral activated \u003cem\u003etrans\u003c/em\u003e-cleavage activity of CRISPR-Cas12a, the method achieves a detection limit of \u0026zwnj;10\u003csup\u003e1\u003c/sup\u003e CFU/mL\u0026zwnj;, with no cross-reactivity against other common food-borne pathogens. This approach employs the fluorophore-labeled DNA reporters that are cleaved by activated Cas12a, allowing for rapid and on-site visualization of detection results. Validation in different food matrices yields satisfactory results, demonstrating robustness against matrix interference. Comparative analysis revealed a \u0026zwnj;10-fold sensitivity improvement\u0026zwnj; over traditional single-primer PCR protocols, attributed to the dual amplification efficiency of designed one-tube nested PCR and the collateral activated cleavage specificity of CRISPR-Cas12a. The portability, rapid visual readout, and ultrasensitive performance of the method enable real-time, on-site screening of \u003cem\u003eSalmonella\u003c/em\u003e in diverse food supply chains, even in resource-limited settings. Its high specificity, robustness against matrix effects, and minimal equipment requirements make it a transformative, user-friendly tool for enhancing global food safety surveillance and preventing outbreaks.\u003c/p\u003e","manuscriptTitle":"One-tube nested PCR mediated CRISPR-Cas12a: A novel performance-enhanced approach for ultrasensitive fluorescent visual screening of Salmonella","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 22:50:32","doi":"10.21203/rs.3.rs-7534316/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"978b6e47-ed9a-476e-b682-bd1e1f4fb2b5","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-05T08:25:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-18 22:50:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7534316","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7534316","identity":"rs-7534316","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}