A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assay

A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assay | 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 A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assay Yunkai Zhang, Yan Xiang, Dengyong Hou, Liben Fang, Shuqi Cai, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5729352/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Mar, 2025 Read the published version in BMC Microbiology → Version 1 posted 4 You are reading this latest preprint version Abstract Dengue Virus (DENV) is a severe life-threatening virus to human, which is the major cause for dengue fever. However, the efficiency of traditional detection methods unable to meet the additional requirements in clinic practice, including the virus isolation method, ELISA, RT-PCR and qRT-PCR and so on. Therefore, a rapid, simple, and accurate diagnostic for DENV is highly desired. In the current study, we developed a novel method for universal DENV detection via introducing recombinase polymerase amplification (RPA) assay and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and associated (Cas) protein 12a (CRISPR/Cas12a) system in one-pot, achieving the extremely sensitivity and specificity for DENV. The whole detection assay can be finished within 40 min without the requirement for sophisticated equipment. The limit of detection (LoD) was 91.7 copies per test. All the recombinant plasmid of these four serotypes of DENV (I to IV) were successfully identified by using present one-pot DENV universal RT-RPA CRISPR/Cas12a detection system. As for specificity, a total of 22 DENV positive samples were successfully identified, and no-cross reactions were observed in 4 other interferes nuclear acid samples. Moreover, we also established the universal DENV RT-RPA-CRISPR/Cas12a- lateral flow dipstick (LFD) platform and all the four serotypes of DENV (I to IV) were successfully identified, reaching the sensitivity of about 250 copies/test. Together, our present method not only provided an alternative approach for universal DENV detection but also gained a novel insight for other virus identification. DENV RPA CRISPR/Cas12a one-pot LFD universal detection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction As a well-known mosquito-borne virus, Dengue virus (DENV) is widespread in tropical and subtropical regions of the world (1). The clinical manifestations of DENV range from mild dengue fever (DF) to severe and fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (2). The DENV is a single-strander positive-sense RNA with the length of 11kb, which belongs to the genus Flavivirus within the family of Flaviviridae . Commonly, there are four known genetically different serotypes according to plaque reduction neutralization test, includes DENV-I, DENV-II, DENV-III, and DENV-IV (3). Moreover, multiple genotypes are identified for each DENV serotype, demonstrating the massive diversity of DENV (4). DENV-I is the most widespread serotype and includes five genotypes. As for distribution, DENV-I was the dominant strains in Southeast Asian countries (Vietnam, Laos, Thailand, and Myanmar), East Asian countries (China, Japan), African region and American region, Malaysia, Australia, Indonesia, and Philippines. DENV-II contains six genotypes, which was common is Southeast Asia, America and Philippines. As for DENV-III, there was five genotypes and distributed in Southeast Asia, the Indian subcontinent, the South Pacific, East Africa, and the Americas. The serotype of DENV-IV contains three genotypes and was common in Malaysia, South Asia and Southeast Asia respectively (5, 6). Over 390 million infections and 25.000 deaths were caused by DENV annually worldwide (7). The majority of pooled mortality rate was caused by DENV-II, followed by DENV-III, DENV-IV, and DENV-1(6). However, the available vaccines or specific therapeutics have not been approved in terms for preventing its rapid emergence and global spread (8, 9). Therefore, an early, simple, and accurate method for DENV universal detection is critical important to implement the preventive measures for DENV infections. Commonly, the traditional detection approaches for DENV include virus isolation, serological, enzyme-linked immunosorbent assay (ELISA), reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (qRT-PCR) have been developed and widely applied to diagnosis of dengue infections (10-12). However, these methods are highly depended on the specific DENV antibodies, sophisticated equipment and well-trained staffs. Therefore, these methods are limited to implement in resource-poor regions by these difficulties. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated (Cas) proteins (CRISPR/Cas) technique has extended for nucleic acid detection, the representing platform includes Cas13a-based SHERLOCK(Specific High Sensitivity Enzymatic Reporter UnLOCKing) (13), Cas12a-based DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) (14), HOLMES (one-hour low-cost multipurpose highly efficient system) (15) and Cas12b-based HOLMESv2 (16). The Cas protein can recognize and cleave the target viral nucleic acid sequence by using specific CRISPR guide RNA (gRNA), and then extensively cleave non-target single-stranded nucleic acids in the system. (14). By using fluorescent dye and quencher-labeled ssDNA reporter a remarkable fluorescence signal can be observed in the presence of the target nuclear acid sequence, while no signal can be detected in the absence of the target nuclear acid sequence (13, 16, 17). As the Cas protein was extremely sensitivity and cannot tolerate even one single base mismatch of the target nuclear acid sequence, attaining super specificity for nuclear acid sequences detection. Moreover, a preamplification assay by isothermal amplification assay was frequently suggested into the CRISPR/Cas system to improve the sensitivity. Near recently, Vikrant Nain and colleagues have designed specific gRNAs for distinguishing the four DENV serotypes by using CRISPR/Cas13-based method (18). However, the performance of these gRNAs was unclear. Therefore, establishing a simple, timely and accurate detection system for universal DENV detection was highly desired. Most of the CRISPR-based systems included pre-amplification and CRISPR/Cas detection two separated major steps, and the amplified-products need to be transferred into CRISPR/Cas system via uncapping operation. Therefore, these platforms suffered from the risk for cross-contamination. In the current study, we firstly developed a system for universal DENV detection via incorporating RT-RPA assay with CRISPR/Cas12a system in one-pot. In this system, the RT-RPA reaction was prepared at the bottom of the tube, while the CRISPR/Cas12a was set in the tube cap. After amplification, the CRISPR/Cas12a detection system was mixed into RT-RPA system with short spin. Therefore, the cross-contamination was totally avoided and realizing the universal detection for four DENV serotypes. Moreover, the corresponding RT-RPA-CRISPR/Cas12a lateral flow dipstick (LFD) platform was established and well-functioned for these four DENV serotypes detection. Our present findings not only provided an alternative approach for universal DENV detection but also demonstrated the potential value of RT-RPA CRISPR/Cas12a one-pot detection system for other virus detection. Materials and methods Cells and Virus Huh7 and A549 cells were grown in complete Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) with penicillin and streptomycin (Gibco, Invitrogen, USA). Aedes albopictus mosquito (C6/36) cells (kindly provided by Prof. Jing An from Capital Medical University, Beijing, China) were cultured in RPMI-1640 medium (Gibco, Invitrogen, USA) supplemented with 10% FBS and antibiotics at 28℃. DENV-II virus (strain Tr1751) was kindly given by Prof. Jing An from Capital Medical University, Beijing, China, which was propagated in C6/36 cells. Viral titers were detected by plaque assay and are shown as plaque-forming units (PFU) per mL. VSV and IAV(H1N1) was described previously (19, 20). Design of the DENV-specific RT-RPA assay primer and Cas12a crRNA According to the highly conserved region of the genomes of all four DENV serotypes, a total of 2 forward and 7 reverse RPA primers DENV-specific primers were designed. The coverage of the RT-RPA primers was examined by using viral RNA samples that extracted from the different reference DENV strains. The Cas12a crRNA was designed by the amplified sequence of the DENV genomic. All the RT-RPA and crRNA were synthesized by Sangon Biotech (Shanghai, China) and listed as follows. The Cas12a crRNA was purified by using Cas12a High Yield crRNA synthesis and purification kit (#31904, ToloBio, Shanghai, China) with the guidance given by the manufacture. The related sequences were listed in Table 1 and Table 2. The one-pot RT-RPA-CRISPR/Cas12a DENV universal detection system. The final volume of the RT-RPA reaction was 10 μL and set at the bottom of the tube, including A Buffer 5.88 μL, F1 (10 μM), primer 0.4 μL, R1 (10 μM) primer 0.4 μL, B Buffer 0.5μL, Nuclease-free water 1.82 μL and Template 1 μL. The whole assay was performed at 40℃for 30min. The amplification product was confirmed by gel electrophoresis. The total volume of the CRISPR/Cas12a was 10 μL and set in tube cap, consisting of 10x HOLMES Buffer 2 μL, LbCas12a 1 μL, HOLMES ssDNA reporter (FAM) 2 μL, crRNA 2 μL and Nuclease-free water 3 μL. After amplification, the CRISPR/Cas12a detection reaction was centrifuged into RPA system for detection at 48℃ for 10 min.. The dynamic FAM fluorescence signals were detected every 30 seconds by using the Applied Biosystems QuanStudio 5 real-time PCR system (QuantStudio 5, ThermoFisher, USA). Specificity and sensitivity of RT-RPA-CRISPR/Cas12a for DENV As for specificity, 22 cell lyases from dengue viruses-infected samples and 4 interfere samples (VSV or IAV- infected A549 and Huh7 cells) were prepared as described previously (19, 21). The RNA was extracted by TRIZOL (Invitrogen, USA), according to the protocol as described (22). The high concentration of synthetic RNA transcript of dengue virus was used as the positive control. Meanwhile, a total of 8 interferes virus samples were collected for specificity detection, including four RNA samples and four DNA samples of sendai virus (SeV), Herpes simplex virus (HSV), Parainfluenza A virus (IAV), and vesicular stomatitis virus (VSV). The recombinant DENV plasmid was served as the positive control (PC) and the nuclease-free water was used as the negative control (NC). Analytical sensitivity testing for DENV RT-RPA-CRISPR/Cas12a assay, the recombinant plasmid serially diluted by two-fold, from 3.125 copies/test to 100 copies/test respectively. Eight replications were performed at each dose. The LoD was analyzed by using Sigmoid function. Detection with test strip using RT-RPA-CRISPR/Cas12a lateral flow dipstick (LFD) platform for DENV universal detection. The total volume of RT-RPA was 10 ul, including A buffer 5.88 μl, Forward primer F1 (10 μM) 0.4 μl, reverse primer R1 (10 μM) 0.4 μl, B buffer 0.5 μl, Template (500 copies/μl) 2 μl, and Nuclease-free water 0.82 μl. The RT-RPA reaction was performed at 38℃ for 30 min. The CRISPR cleavage reaction system consists of 10x HOLMES Buffer 2μl, LbCas12a (10 μM) 1μl, FAM-T7-Biotin Reporter (10 μM) 0.5μl, -crRNA2 (10 μM) 2μl, Nuclease-free water 4.5μl. The RT-RPA reaction was set as the bottom of the tube, while the CRISPR/Cas12a detection system was prepared in the tube cap and mixed into the RT-RPA reaction with short spin and performed at 48℃ for 10min. After that, 5μl of reaction mixture was added to 95 μl Nuclease-free water and then the colloidal gold test strip was inserted for detection (31203, TOLO biotech, China), with the results observed within 10 min at room temperature. After incubation for 2–5 min, the test line (T-line) and control line (C-line) appeared, and the results were obtained based on the color of the test strip. In the absence of the target nuclear acid sequence, the C line was visible, and the T line was invisible. With a target, the ssDNA reporter molecule was trans-cleaved by the activated Cas12a protein, and the FITC and biotin molecules were separated. The FITC–anti-FITC anti- body–AuNP complexes were then captured by the goat anti-rabbit IgG antibody on the T line, which gave a positive readout. Due to the incomplete trans-cleavage of the ssDNA reporter molecule, both cleaved and intact ssDNA reporters were present in a positive assay, resulting in red bands at both the T line and the C line. Results The workflow of one-pot RPA CRISPR/Cas12a method for DENV detection. The workflow of one-pot RPA CRISPR/Cas12a method for DENV detection was shown in Figure 1. Firstly, the nuclear acid of DENV was isolated from the positive-infected samples and used as the template. Then, the RT-RPA assay was used to amplify the target nuclear acid sequence by using a universal primer according to the conserved region of DENV four genetically different serotypes and the RT-RPA amplification assay was set at the bottom of the tube. Then, the CRISPR/Cas12a detection reaction was prepared in tube cap and centrifuged into RT-RPA amplification for detection with a FAM-labeled single-stranded DNA reporter. The CRISPR/Cas12a system could be activated and cleaved the FAM-labelled reporter to lead to a fluorometric response once recognizing target nuclear acid sequence by specific crRNA. The whole detection process achieved contamination-free without additional uncapping step and universal DENV detection. The total detection time of present method was 40 min, including 30 min for RT-RPA amplification and 10 min for CRISPR/Cas12a detection. Moreover, the corresponding RT-RPA-CRISPR/Cas12a lateral flow dipstick (LFD) platform was established and well-functioned for universal DENV detection. The RPA primer and crRNA selection According to the conserved nucleic acid sequence of DENV, a total of 2 forward and 7 reverse RPA primers were synthesized. As shown in Figure 2, the product by using primer F1/R1 showed the best quality than that of other primers. Therefore, the primer F1/R1 was chosen for RPA amplification. According to the amplified fragment, a number of 4 crRNAs were designed. Taking the threshold time and fluorescence intensity in consideration, the crRNA2 presented the best performance (Figure 3). Therefore, the crRNA2 was chosen for detection. Optimized the one-pot RT-RPA CRISPR/Cas12a detection reaction. To improve the efficiency of the detection, the concentrations of Cas12a and crRNA were optimized. The concentration of Cas12a and crRNA were diluted into 125 nM, 250 nM, 500 nM and crRNA were diluted into 125 nM, 250 nM, 500 nM and 1000 nM respectively. As shown in Figure 4A, the fluorescence intensity of the detection reaction reached the highest value by using the Cas12a in 500 nM and crRNA in 1000 nM. Temperature is key element for the detection reaction. Here, the reaction temperature was ranged from 38℃ to 42℃by 2℃ increment. In the temperature of 38℃, the fluorescence intensity of the detection reaction attained the highest value. Then, the temperature was optimized in 38℃ (Figure 4B). Sensitivity and specificity analysis of one-pot RT-RPA CRISPR/Cas12a detection reaction. To determine the sensitivity of the detection reaction, the limit of detection (LoD) was verified by serially diluting the recombinant plasmid by two-fold, from 3.125 copies/test to 100 copies/test respectively. As shown in Figure 5, the LoD of present reaction was achieved 91.7 copies per test at the probability of 95%. As for specificity, a total of 26 virus samples were collected and used for detection, including 22 DENV samples and 4 non-DENV samples. All the DENV-positive samples were accurately identified, and no cross-reaction were observed, indicated the 100% specificity of present detection system for DENV (Figure 6A). Moreover, a total of 8 interferes virus samples were collected for detection, including four RNA samples and four DNA samples of sendai virus (SeV), Herpes simplex virus (HSV), Parainfluenza A virus (IAV), and vesicular stomatitis virus (VSV). The recombinant DENV plasmid was served as the positive control (PC) and the nuclease-free water was used as the negative control (NC). As shown in Figure 6B, only the PC induced a remarkable fluorescence intensity, and no remarkable signal were collected from the interfere samples. All these data suggested that the present system was specificity for DENV and no-cross reaction were identified. Validation of the one-pot RT-RPA CRISPR/Cas12a DENV detection system. To examine the effectiveness of present platform for universal DENV detection, the recombinant plasmids that containing the corresponding nuclear acid sequence of DENV-I, DENV-II, DENV-III, and DENV-IV were synthesized and used for detection. As shown in Figure 7A, all these four serotypes of DENV were successfully identified by using present CRISPR-based detection system. Establish and validation of the RT-RPA CRISPR/Cas12a-LFD DENV detection system. Meanwhile, we also constructed the DENV RT-RPA-CRISPR/Cas12a-LFD platform and used for detecting the four recombinant plasmid of DENV. As shown in Figure 7B, all the four serotypes of DENV were identified by using corresponding RT-RPA-CRISPR/Cas12a-colloidal gold test strip platform respectively (Figure 7B). Meanwhile, the FAM-T7-Biotin Reporter that used in the reaction was optimized by serially adjusting by two-fold from 200 nM to 25 nM respectively. As shown in Figure 7C, the efficiency of the test strip showed no significant difference by using the ssDNA reporter in different dose. For saving costs, the dose of the ssDNA reporter was determined in 25 nM for the test strip assay. To validate the sensitivity of the present test strip assay, the cultured DENV-II virus as indicated above were used for detection. The template of DENV-II nuclear acid samples was diluted into 2000 copies/test, 1000 copies/test, 500 copies/test, 250 copies/test, and 125 copies/test respectively. Four test replications were performed as each dose. As shown in Figure 7D, all the four replications were identified by using the template in the dose of 2000 copies/test, 1000 copies/test and 500 copies/test, while three for 250 copies/test and two for 125 copies/test respectively. All these results demonstrated that the present RT-RPA CRISPR/Cas12a-LFD was well-functioned for universal DENV detection. Discussion DENV is a deadly virus that deeply threaten the human health and life quantity, over 4 billion people are living at a risk of dengue infection (23). However, the available therapy or vaccine are not approved at present. Therefore, an early detection method for DENV is essential to prevent its pandemic and optimize the coping strategies. With the rapid development of molecule detection techniques, the PCR-based method is widely applied as a routine assay for DENV diagnosis and offered high sensitivity (24-26). However, this method was limited by serval shortcomings, such as the requirement for expensive thermal cycling instrumentation, technical expertise, and prolonged testing times. Compared with PCR-based method, several reverse transcription recombinase polymerase amplifications (RT-RPA)-assist methods have been used for DENV detection and does not need thermal cycling instrumentation, which can performed as at constant temperature with limited testing time (27-29).However, the non-specific product during the amplification cannot totally avoided, resulting the high rate of false-positive results (30, 31). For this issue, Feng Zhang ‘s group have firstly established a SHERLOCK platform to detect DENV, offering high sensitivity to 2 aM. (13). However, this platform included two separated steps can only identify strain 1 or 3 of DENV, not available for the strain 2 or strain 4 of DENV and avoiding cross-contamination. Moreover, Vikrant Nain and colleagues have employed DENV-specific CRISPR-Cas13a gRNAs for the conserved and variable genomic regions among four DENV serotypes using bioinformatics tools, which can be used in diagnosing the dengue virus and its serotypes for in vitro (18). However, the performance of these gRNAs needs to be further validated. Huangxian Ju et.al have developed an electrochemical method based on CRISPR/Cas13a-assisted catalytic hairpin assembly (CHA) system for DENV detection, achieving the detection limit of 0.78 fM for detecting DENV type 1(32). However, this method requires high-cost electrochemical biosensor, professional technicians and cannot discriminate other type of DENV. In addition, Qianfeng Xia and colleagues have developed a bienzyme method for DENV detection based on Cas13a and Cas12a. (33). However, this platform can only detect DENV type 1 and not available for other types. In China, all the four serotypes and various genotypes of DENV strains have been identified and the incidence of DENV infection is increasingly serious in recent years (34, 35). Due to the multiple serotypes and genotypes of the DENV, most of the detection platform can only detected the dominant serotypes on specific regions and the universal DENV detection platform remains unknown by technique limitations. In the current study, we firstly developed a universal DENV detection system via combining RT-RPA amplification and CRISPR/Cas12a assay in one-pot. According to the multiple sequence alignment of four DENV serotypes, a pair of degenerate primers was selected and used for amplifying the target sequence that including all variations of these four DENV serotypes. Then, a Cas12a crRNA was used to recognize the target sequence. Importantly, the one-pot DENV universal detection system was well-functioned and successfully identified four recombinant plasmids that containing the conserved region of DENV-I to DENV-IV respectively. Moreover, the corresponding RT-RPA CRISPR/Cas12a-LFD platform was also established and showed a well performance by detecting the recombinant plasmid. As the LFD was cheap and convenient, our present DENV detection system has the potential value to extend for resources-limited regions, especially for developing countries and contributed to developing DENV Point-of-care Testing (POCT) and in vitro diagnostic (IVD) products. Moreover, the whole assay can be finished in 40 min without the requirement for sophisticated equipment and cross operations. Moreover, there was no cross-reaction with other RNA viruses. Therefore, the RT-RPA CRISPR/Cas12a platform was extremely specificity for DENV, avoiding the likelihood of misdiagnosis. In addition, there was no need to uncapping procedures during the detection. Therefore, the current one-pot RT-RPA CRISPR/Cas12a assay yielded outstanding advantages for universal DENV detection, achieving rapid, accurate, simple and contamination-free properties. Furthermore, all the component that can be pre-prepared by lyophilized powder and the whole assay can be performed on a simple thermal heater, even at room temperature. Therefore, the RPA CRISPR/Cas12a platform has the potential value to extend in resource-poor settings. Together, our present findings not only offered a promising available approach for universal DENV detection but also provided novel insight for developing the methods for identification other mankind viruses. Conclusion In this study, we introduced the RT-RPA assay and CRISPR/Cas12a system in one-pot for universal DENV detection and established the corresponding RT-RPA CRISPR/Cas12a-LFD system. The RT-RPA CRISPR/Cas12a method presented high specificity and sensitivity for DENV without false-positive results, achieving rapid, accurate, simple and contamination-free properties. Declarations Author Contributions Y.Z., X.L. and M.R. supervised and secured funding for the project; Y.Z., X.L. and M.R. conceived the study, designed experiments, analyzed data, and revised the manuscript; Y.Z., Y.X. and D.H. designed experiments, performed experiments, analyzed data, interpreted data and drafted the manuscript. L.F., S.C., J.Z., Y.W., Y.J., B.L., J.B., Y.D., J.F. and S.C. performed experiments and collected data. Funding This work was supported by the National Key Research & Development Program of China (2023YFC2307302), the National Natural Science Foundation of China (32400727), the program of Shanghai outstanding academic leader in public health subject (GWVI-11.2-XD29), the experimental animal program sponsored by the Science and Technology Commission of Shanghai Municipality (23141902300), and Natural Science Foundation of Shanghai (24ZR1481100). Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Acknowledgements Not applicable. References Harapan H, Michie A, Sasmono RT, Imrie A. Dengue: A Minireview. Viruses. 2020;12(8). Kraivong R, Punyadee N, Liszewski MK, Atkinson JP, Avirutnan P. 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Leon F, Pinchon E, Mayran C, Daynès A, Morvan F, Molès JP, et al. Magnetic Field-Enhanced Agglutination Readout Combined With Isothermal Reverse Transcription Recombinase Polymerase Amplification for Rapid and Sensitive Molecular Detection of Dengue Virus. Front Chem. 2021;9:817246. Abd El Wahed A, Patel P, Faye O, Thaloengsok S, Heidenreich D, Matangkasombut P, et al. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection. PLoS One. 2015;10(6):e0129682. Jang M, Kim S. Inhibition of Non-specific Amplification in Loop-Mediated Isothermal Amplification via Tetramethylammonium Chloride. Biochip J. 2022;16(3):326-33. Gao X, Sun B, Guan Y. Pullulan reduces the non-specific amplification of loop-mediated isothermal amplification (LAMP). Anal Bioanal Chem. 2019;411(6):1211-8. Wang J, Xia Q, Wu J, Lin Y, Ju H. A sensitive electrochemical method for rapid detection of dengue virus by CRISPR/Cas13a-assisted catalytic hairpin assembly. Anal Chim Acta. 2021;1187:339131. Tian G, Tan J, Liu B, Xiao M, Xia Q. Field-deployable viral diagnostic tools for dengue virus based on Cas13a and Cas12a. Anal Chim Acta. 2024;1316:342838. Yang L, Chen Y, Yan H, Zhang P, Xu X, Tang B, et al. A survey of the 2014 dengue fever epidemic in Guangzhou, China. Emerg Microbes Infect. 2015;4(9):e57. Wu T, Wu Z, Li YP. Dengue fever and dengue virus in the People's Republic of China. Reviews in medical virology. 2022;32(1):e2245. Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.pdf Table2.pdf Cite Share Download PDF Status: Published Journal Publication published 25 Mar, 2025 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 16 Jan, 2025 Editor assigned by journal 03 Jan, 2025 Submission checks completed at journal 03 Jan, 2025 First submitted to journal 29 Dec, 2024 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5729352","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":397332453,"identity":"df71e819-ce4d-4174-8b01-24060c03aad8","order_by":0,"name":"Yunkai Zhang","email":"","orcid":"","institution":"Naval Medical Center, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yunkai","middleName":"","lastName":"Zhang","suffix":""},{"id":397332454,"identity":"254dbdfe-c39a-4f32-a760-27518ec44399","order_by":1,"name":"Yan Xiang","email":"","orcid":"","institution":"Department of Pathogen Biology, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Xiang","suffix":""},{"id":397332455,"identity":"fc49a395-c415-4dc3-bf2a-96757b54c902","order_by":2,"name":"Dengyong Hou","email":"","orcid":"","institution":"Naval Medical Center, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dengyong","middleName":"","lastName":"Hou","suffix":""},{"id":397332456,"identity":"abc3cfe8-791a-414e-a0dc-3232fb230860","order_by":3,"name":"Liben Fang","email":"","orcid":"","institution":"Naval Medical Center, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liben","middleName":"","lastName":"Fang","suffix":""},{"id":397332457,"identity":"458f1795-6d71-4941-a71f-cbb86d0fb8ad","order_by":4,"name":"Shuqi Cai","email":"","orcid":"","institution":"Naval Medical Center, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuqi","middleName":"","lastName":"Cai","suffix":""},{"id":397332458,"identity":"a11f90b3-4cd7-457e-bf55-a499eca3a036","order_by":5,"name":"Jianping Zhang","email":"","orcid":"","institution":"Naval Medical Center, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianping","middleName":"","lastName":"Zhang","suffix":""},{"id":397332459,"identity":"74c7ac04-dcbd-4352-abfc-0f151a74006d","order_by":6,"name":"Yujia Wang","email":"","orcid":"","institution":"Department of Immunology, Center for Immunotherapy, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yujia","middleName":"","lastName":"Wang","suffix":""},{"id":397332460,"identity":"3d61273e-e715-47f1-8eba-45b62d6af587","order_by":7,"name":"Yuyu Jiang","email":"","orcid":"","institution":"Department of Pathogen Biology, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuyu","middleName":"","lastName":"Jiang","suffix":""},{"id":397332461,"identity":"12c92fe6-92f0-4ac0-b6c9-49d92a503343","order_by":8,"name":"Bin Liu","email":"","orcid":"","institution":"Naval Medical Center, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Liu","suffix":""},{"id":397332462,"identity":"ef83a841-6654-4267-a9d8-5eada7b0fc79","order_by":9,"name":"Jie Bai","email":"","orcid":"","institution":"Department of Pathogen Biology, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Bai","suffix":""},{"id":397332463,"identity":"17ef5588-f876-4da6-b6d1-f8176dabc2ec","order_by":10,"name":"Yue Ding","email":"","orcid":"","institution":"Department of Pathogen Biology, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Ding","suffix":""},{"id":397332464,"identity":"c9702e34-ed49-4b57-8e9c-a75114f3bc48","order_by":11,"name":"Jingjing Fang","email":"","orcid":"","institution":"Naval Medical Center, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Fang","suffix":""},{"id":397332465,"identity":"3f3bce95-051f-42db-89de-d7236f120d37","order_by":12,"name":"Shuanghong Chen","email":"","orcid":"","institution":"Naval Medical Center, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuanghong","middleName":"","lastName":"Chen","suffix":""},{"id":397332466,"identity":"4fd4476a-3dc1-4748-a5e1-3997028ea2f3","order_by":13,"name":"Xingguang Liu","email":"","orcid":"","institution":"National Key Laboratory of Immunity \u0026 Inflammation, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xingguang","middleName":"","lastName":"Liu","suffix":""},{"id":397332467,"identity":"81978df7-0a0b-4896-a6ff-6415e55e13cb","order_by":14,"name":"Xiaomeng Ren","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYLCCBAjF+ADKNyBaCzNMKRFaoIBNgigt5u29xyQe1NhE80u3X6v8mbMtsYG9eZsEQ80dnFpkzpxLNkg4lpY7c86Zstu8224nNvAcK5NgOPYMpxYJiRzDBwlsh3M33MhJu80I0iKRYybB2HAYtxb5NwYHEv4dzt0P1FL4E6RF/g0BLRI8hg8S24C2SKQfYwA7TIKHgBaeHGODxL603Bk3cpilgVqM23jSii0SjuHRwn7GTPLHN5vc/hnpDz8CHSbbz354440PNbi1IAEeSHSwgYgEYjQwMLA/IE7dKBgFo2AUjDgAAE4aV/o0ZEazAAAAAElFTkSuQmCC","orcid":"","institution":"Naval Medical Center, Naval Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaomeng","middleName":"","lastName":"Ren","suffix":""}],"badges":[],"createdAt":"2024-12-29 09:53:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5729352/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5729352/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-025-03882-z","type":"published","date":"2025-03-25T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73025772,"identity":"8a5c46d5-7f15-48c1-83f4-a40f15d8d4b1","added_by":"auto","created_at":"2025-01-06 05:00:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1749431,"visible":true,"origin":"","legend":"\u003cp\u003eSchema illustration of one-pot RPA CRISPR/Cas12a workflow for DENV virus detection.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5729352/v1/ab106694d9156df1971a8bd4.png"},{"id":73025771,"identity":"285fa4fe-9d13-41a7-af81-379f327deb1d","added_by":"auto","created_at":"2025-01-06 05:00:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":394326,"visible":true,"origin":"","legend":"\u003cp\u003eSelection of the RPA primer for one-pot RPA CRISPR/Cas12a system. Two of forward primer (F1 and F2) and six reverse primers were individually combined. The products were examined by using 2% agarose gel.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5729352/v1/3b0282b1b8d32e7895bc9a9a.png"},{"id":73026440,"identity":"53b6b0ea-358e-4081-b1c9-f64a7accc544","added_by":"auto","created_at":"2025-01-06 05:08:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":431189,"visible":true,"origin":"","legend":"\u003cp\u003eSelection of the Cas12a crRNA for one-pot RPA CRISPR/Cas12a system. The detection results of four crRNAs were respectively shown in crRNA1(\u003cstrong\u003eA\u003c/strong\u003e), crRNA2(\u003cstrong\u003eB\u003c/strong\u003e),\u003c/p\u003e\n\u003cp\u003eCrRNA3(\u003cstrong\u003eC\u003c/strong\u003e) and crRNA4(\u003cstrong\u003eD\u003c/strong\u003e). The purple curve represented the crRNA signal, and the green one represented the negative control.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5729352/v1/f96547b46e24fab3404895a6.png"},{"id":73025774,"identity":"4bdcfad9-4c5d-48aa-a001-2d92bb802da5","added_by":"auto","created_at":"2025-01-06 05:00:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":194373,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of the one-pot RPA CRISPR/Cas12a system for DENV detection. (\u003cstrong\u003eA\u003c/strong\u003e) Optimization of the concentrations of Cas12a and crRNA. Cas12a was diluted in 125 nM,250 nM, and 500 nM, while crRNA was diluted in 125 nM,250 nM, 500 nM and 1000 nM. (\u003cstrong\u003eB\u003c/strong\u003e) Optimization of the incubation temperature for one-pot RPA CRISPR/Cas12a system. The reaction temperature was set at 38℃, 40℃ and 42 ℃ respectively. Three replications for each reaction.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5729352/v1/7c672d7081b7b8d4366b1f44.png"},{"id":73025770,"identity":"e4b4a6cd-e8c5-4d28-bfd1-caeabec760c8","added_by":"auto","created_at":"2025-01-06 05:00:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":80787,"visible":true,"origin":"","legend":"\u003cp\u003eThe analysis of LoD for DENV one-pot RPA CRISPR/Cas12a detection system. The LoD of present system achieved 91.7 copies/test at the probability of 95%.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5729352/v1/75c16b8dd4d9a4b5f030db38.png"},{"id":73025775,"identity":"7c0ba887-fc8b-4243-b539-ddc30057639e","added_by":"auto","created_at":"2025-01-06 05:00:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":466856,"visible":true,"origin":"","legend":"\u003cp\u003eThe specificity analysis of DENV one-pot RPA CRISPR/Cas12a detection system. A. The nucleic acid samples were from Huh7 or A549 cells infected with DENV, VSV and IAV. B. The RNA and DNA samples of sendai virus (SeV), Herpes simplex virus (HSV), Parainfluenza A virus (IAV), and vesicular stomatitis virus (VSV) were used for examining the specificity of present system, three technical replications for each biological sample.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5729352/v1/390df1928ab2e95f7e442793.png"},{"id":73025776,"identity":"cefea3fc-2317-4168-a18e-b8d68d9d9da8","added_by":"auto","created_at":"2025-01-06 05:00:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6052841,"visible":true,"origin":"","legend":"\u003cp\u003eEstablishment of the RT-RPA CRISPR/Cas12a-LFD system. A. The recombinant plasmid of DENV-I, DENV-II, DENV-III, and DENV-IV were identified by using the one-pot universal DENV RT-RPA CRISPR/Cas12a detection system. B. The recombinant plasmid of DENV-I, DENV-II, DENV-III, and DENV-IV were identified by using the DENV RT-RPA CRISPR/Cas12a-LFD detection system. C. The ssDNA reporter that used in DENV RT-RPA CRISPR/Cas12a-LFD detection system was optimized via serially diluting by two-fold, from 200 nM to 25 nM respectively. D. Sensitivity analysis of the DENV RT-RPA CRISPR/Cas12a-LFD detection system. The template was diluted into 2000 copies/test, 1000 copies/test, 500 copies/test, 250 copies/test and 125 copies/test respectively, n = 4 for each group.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5729352/v1/708918088136f5d8236811ab.png"},{"id":79605131,"identity":"a5b3c37a-0c0a-4b71-8c3e-439cbf97ef32","added_by":"auto","created_at":"2025-03-31 16:10:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9096799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5729352/v1/62a330a0-3617-4bc0-a7b2-b34dd7553b81.pdf"},{"id":73025768,"identity":"0a3362ea-f94d-400e-9109-3ef487ab705b","added_by":"auto","created_at":"2025-01-06 05:00:56","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":87380,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5729352/v1/f18313c89c1f4a9516c38662.pdf"},{"id":73026439,"identity":"06b5924a-6016-426a-ab9a-357b040d9b26","added_by":"auto","created_at":"2025-01-06 05:08:56","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":86511,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5729352/v1/f64597edf75e922a8d948813.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assay","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs a well-known mosquito-borne virus, Dengue virus (DENV) is widespread in tropical and subtropical regions of the world (1). The clinical manifestations of DENV range from mild dengue fever (DF) to severe and fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (2). The DENV is a single-strander positive-sense RNA with the length of 11kb, which belongs to the genus \u003cem\u003eFlavivirus\u003c/em\u003e within the family of \u003cem\u003eFlaviviridae\u003c/em\u003e. Commonly, there are four known genetically different serotypes according to plaque reduction neutralization test, includes DENV-I, DENV-II, DENV-III, and DENV-IV (3). Moreover, multiple genotypes are identified for each DENV serotype, demonstrating the massive diversity of DENV (4). DENV-I is the most widespread serotype and includes five genotypes. As for distribution, DENV-I was the dominant strains in Southeast Asian countries (Vietnam, Laos, Thailand, and Myanmar), East Asian countries (China, Japan), African region and American region, Malaysia, Australia, Indonesia, and Philippines. DENV-II contains six genotypes, which was common is Southeast Asia, America and Philippines. As for DENV-III, there was five genotypes and distributed in Southeast Asia, the Indian subcontinent, the South Pacific, East Africa, and the Americas. The serotype of DENV-IV contains three genotypes and was common in Malaysia, South Asia and Southeast Asia respectively (5, 6).\u003c/p\u003e\n\u003cp\u003eOver 390 million infections and 25.000 deaths were caused by DENV annually worldwide (7). The majority of pooled mortality rate was caused by DENV-II, followed by DENV-III, DENV-IV, and DENV-1(6). However, the available vaccines or specific therapeutics have not been approved in terms for preventing its rapid emergence and global spread (8, 9). Therefore, an early, simple, and accurate method for DENV universal detection is critical important to implement the preventive measures for DENV infections.\u003c/p\u003e\n\u003cp\u003eCommonly, the traditional detection approaches for DENV include virus isolation, serological, enzyme-linked immunosorbent assay (ELISA), reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (qRT-PCR) have been developed and widely applied to diagnosis of dengue infections (10-12). However, these methods are highly depended on the specific DENV antibodies, sophisticated equipment and well-trained staffs. Therefore, these methods are limited to implement in resource-poor regions by these difficulties.\u003c/p\u003e\n\u003cp\u003eClustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated (Cas) proteins (CRISPR/Cas) technique has extended for nucleic acid detection, the representing platform includes Cas13a-based SHERLOCK(Specific High Sensitivity Enzymatic Reporter UnLOCKing) (13), Cas12a-based DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) (14), HOLMES (one-hour low-cost multipurpose highly efficient system) (15) and Cas12b-based HOLMESv2 (16). The Cas protein can recognize and cleave the target viral nucleic acid sequence by using specific CRISPR guide RNA (gRNA), and then extensively cleave non-target single-stranded nucleic acids in the system. (14). By using fluorescent dye and quencher-labeled ssDNA reporter a remarkable fluorescence signal can be observed in the presence of the target nuclear acid sequence, while no signal can be detected in the absence of the target nuclear acid sequence (13, 16, 17). As the Cas protein was extremely sensitivity and cannot tolerate even one single base mismatch of the target nuclear acid sequence, attaining super specificity for nuclear acid sequences detection. Moreover, a preamplification assay by isothermal amplification assay was frequently suggested into the CRISPR/Cas system to improve the sensitivity. Near recently, Vikrant Nain and colleagues have designed specific gRNAs for distinguishing the four DENV serotypes by using CRISPR/Cas13-based method (18). However, the performance of these gRNAs was unclear. Therefore, establishing a simple, timely and accurate detection system for universal DENV detection was highly desired.\u003c/p\u003e\n\u003cp\u003eMost of the CRISPR-based systems included pre-amplification and CRISPR/Cas detection two separated major steps, and the amplified-products need to be transferred into CRISPR/Cas system via uncapping operation. Therefore, these platforms suffered from the risk for cross-contamination. In the current study, we firstly developed a system for universal DENV detection via incorporating RT-RPA assay with CRISPR/Cas12a system in one-pot. In this system, the RT-RPA reaction was prepared at the bottom of the tube, while the CRISPR/Cas12a was set in the tube cap. After amplification, the CRISPR/Cas12a detection system was mixed into RT-RPA system with short spin. Therefore, the cross-contamination was totally avoided and realizing the universal detection for four DENV serotypes. Moreover, the corresponding RT-RPA-CRISPR/Cas12a lateral flow dipstick (LFD) platform was established and well-functioned for these four DENV serotypes detection. Our present findings not only provided an alternative approach for universal DENV detection but also demonstrated the potential value of RT-RPA CRISPR/Cas12a one-pot detection system for other virus detection.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch2\u003eCells and Virus\u003c/h2\u003e\n\u003cp\u003eHuh7 and A549 cells were grown in complete Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) containing 10% fetal bovine serum (FBS) with penicillin and streptomycin (Gibco, Invitrogen, USA). \u003cem\u003eAedes albopictus\u003c/em\u003e mosquito (C6/36) cells (kindly provided by Prof. Jing An from Capital Medical University, Beijing, China) were cultured in RPMI-1640 medium (Gibco, Invitrogen, USA) supplemented with 10% FBS and antibiotics at 28℃.\u003c/p\u003e\n\u003cp\u003eDENV-II virus (strain Tr1751) was kindly given by Prof. Jing An from Capital Medical University, Beijing, China, which was propagated in C6/36 cells. Viral titers were detected by plaque assay and are shown as plaque-forming units (PFU) per mL. VSV and IAV(H1N1) was described previously (19, 20).\u003c/p\u003e\n\u003ch2\u003eDesign of the DENV-specific RT-RPA assay primer and Cas12a crRNA\u003c/h2\u003e\n\u003cp\u003eAccording to the highly conserved region of the genomes of all four DENV serotypes, a total of 2 forward and 7 reverse RPA primers DENV-specific primers were designed. The coverage of the RT-RPA primers was examined by using viral RNA samples that extracted from the different reference DENV strains. The Cas12a crRNA was designed by the amplified sequence of the DENV genomic. All the RT-RPA and crRNA were synthesized by Sangon Biotech (Shanghai, China) and listed as follows. The Cas12a crRNA was purified by using Cas12a High Yield crRNA synthesis and purification kit (#31904, ToloBio, Shanghai, China) with the guidance given by the manufacture. The related sequences were listed in Table 1 and Table 2. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eThe one-pot RT-RPA-CRISPR/Cas12a DENV universal detection system.\u003c/h2\u003e\n\u003cp\u003eThe final volume of the RT-RPA reaction was 10 \u0026mu;L and set at the bottom of the tube, including A Buffer 5.88 \u0026mu;L, F1 (10 \u0026mu;M), primer 0.4 \u0026mu;L, R1 (10 \u0026mu;M) primer 0.4 \u0026mu;L, B Buffer\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;0.5\u0026mu;L, Nuclease-free water 1.82 \u0026mu;L and Template 1 \u0026mu;L. The whole assay was performed at 40℃for 30min. The amplification product was confirmed by gel electrophoresis.\u003c/p\u003e\n\u003cp\u003eThe total volume of the CRISPR/Cas12a was 10 \u0026mu;L and set in tube cap, consisting of\u003c/p\u003e\n\u003cp\u003e10x HOLMES Buffer\u0026nbsp; 2 \u0026mu;L, LbCas12a 1 \u0026mu;L, HOLMES ssDNA reporter (FAM) 2 \u0026mu;L, crRNA\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;2 \u0026mu;L and Nuclease-free water 3 \u0026mu;L. After amplification, the CRISPR/Cas12a detection reaction was centrifuged into RPA system for detection at 48℃\u0026nbsp;for 10 min.. The dynamic FAM fluorescence signals were detected every 30 seconds by using the Applied Biosystems QuanStudio 5 real-time PCR system (QuantStudio 5, ThermoFisher, USA).\u003c/p\u003e\n\u003ch2\u003eSpecificity and sensitivity of RT-RPA-CRISPR/Cas12a for DENV\u003c/h2\u003e\n\u003cp\u003eAs for specificity, 22 cell lyases from dengue viruses-infected samples and 4 interfere samples (VSV or IAV- infected A549 and Huh7 cells) were prepared as described previously (19, 21). The RNA was extracted by TRIZOL (Invitrogen, USA), according to the protocol as described (22). The high concentration of synthetic RNA transcript of dengue virus was used as the positive control. Meanwhile, a total of 8 interferes virus samples were collected for specificity detection, including four RNA samples and four DNA samples of sendai virus (SeV), Herpes simplex virus (HSV), Parainfluenza A virus (IAV), and vesicular stomatitis virus (VSV). The recombinant DENV plasmid was served as the positive control (PC) and the nuclease-free water was used as the negative control (NC).\u003c/p\u003e\n\u003cp\u003eAnalytical sensitivity testing for DENV RT-RPA-CRISPR/Cas12a assay, the recombinant plasmid serially diluted by two-fold, from 3.125 copies/test to 100 copies/test respectively. Eight replications were performed at each dose. The LoD was analyzed by using Sigmoid function.\u003c/p\u003e\n\u003ch2\u003eDetection with test strip using RT-RPA-CRISPR/Cas12a lateral flow dipstick (LFD) platform for DENV universal detection.\u003c/h2\u003e\n\u003cp\u003eThe total volume of RT-RPA was 10 ul, including A buffer 5.88 \u0026mu;l, Forward primer F1 (10 \u0026mu;M) 0.4 \u0026mu;l, reverse primer R1 (10 \u0026mu;M) 0.4 \u0026mu;l, B buffer 0.5 \u0026mu;l, Template (500 copies/\u0026mu;l) 2 \u0026mu;l, and Nuclease-free water 0.82 \u0026mu;l. The RT-RPA reaction was performed at 38℃ for 30 min. The CRISPR cleavage reaction system consists of 10x HOLMES Buffer 2\u0026mu;l, LbCas12a (10 \u0026mu;M) 1\u0026mu;l, FAM-T7-Biotin Reporter (10 \u0026mu;M) 0.5\u0026mu;l, -crRNA2 (10 \u0026mu;M) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;2\u0026mu;l, Nuclease-free water \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 4.5\u0026mu;l. The RT-RPA reaction was set as the bottom of the tube, while the CRISPR/Cas12a detection system was prepared in the tube cap and mixed into the RT-RPA reaction with short spin and performed at 48℃ for 10min. After that, 5\u0026mu;l of reaction mixture was added to 95 \u0026mu;l Nuclease-free water and then the colloidal gold test strip was inserted for detection (31203, TOLO biotech, China), with the results observed within 10 min at room temperature. After incubation for 2\u0026ndash;5 min, the test line (T-line) and control line (C-line) appeared, and the results were obtained based on the color of the test strip. In the absence of the target nuclear acid sequence, the C line was visible, and the T line was invisible. With a target, the ssDNA reporter molecule was trans-cleaved by the activated Cas12a protein, and the FITC and biotin molecules were separated. The FITC\u0026ndash;anti-FITC anti- body\u0026ndash;AuNP complexes were then captured by the goat anti-rabbit IgG antibody on the T line, which gave a positive readout. Due to the incomplete trans-cleavage of the ssDNA reporter molecule, both cleaved and intact ssDNA reporters were present in a positive assay, resulting in red bands at both the T line and the C line.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eThe workflow of one-pot RPA CRISPR/Cas12a method for DENV detection.\u003c/h2\u003e\n\u003cp\u003eThe workflow of one-pot RPA CRISPR/Cas12a method for DENV detection was shown in Figure 1. Firstly, the nuclear acid of DENV was isolated from the positive-infected samples and used as the template. Then, the RT-RPA assay was used to amplify the target nuclear acid sequence by using a universal primer according to the conserved region of DENV four genetically different serotypes and the RT-RPA amplification assay was set at the bottom of the tube. Then, the CRISPR/Cas12a detection reaction was prepared in tube cap and centrifuged into RT-RPA amplification for detection with a FAM-labeled single-stranded DNA reporter. The CRISPR/Cas12a system could be activated and cleaved the FAM-labelled reporter to lead to a fluorometric response once recognizing target nuclear acid sequence by specific crRNA. The whole detection process achieved contamination-free without additional uncapping step and universal DENV detection. The total detection time of present method was 40 min, including 30 min for RT-RPA amplification and 10 min for CRISPR/Cas12a detection. Moreover, the corresponding RT-RPA-CRISPR/Cas12a lateral flow dipstick (LFD) platform was established and well-functioned for universal DENV detection.\u003c/p\u003e\n\u003ch2\u003eThe RPA primer and crRNA selection\u003c/h2\u003e\n\u003cp\u003eAccording to the conserved nucleic acid sequence of DENV, a total of 2 forward and 7 reverse RPA primers were synthesized. As shown in Figure 2, the product by using primer F1/R1 showed the best quality than that of other primers. Therefore, the primer F1/R1 was chosen for RPA amplification. According to the amplified fragment, a number of 4 crRNAs were designed. Taking the threshold time and fluorescence intensity in consideration, the crRNA2 presented the best performance (Figure 3). Therefore, the crRNA2 was chosen for detection.\u003c/p\u003e\n\u003ch2\u003eOptimized the one-pot RT-RPA CRISPR/Cas12a detection reaction.\u003c/h2\u003e\n\u003cp\u003eTo improve the efficiency of the detection, the concentrations of Cas12a and crRNA were optimized. The concentration of Cas12a and crRNA were diluted into 125 nM, 250 nM, 500 nM and crRNA were diluted into 125 nM, 250 nM, 500 nM and 1000 nM respectively. As shown in Figure 4A, the fluorescence intensity of the detection reaction reached the highest value by using the Cas12a in 500 nM and crRNA in 1000 nM.\u003c/p\u003e\n\u003cp\u003eTemperature is key element for the detection reaction. Here, the reaction temperature was ranged from 38℃ to 42℃by 2℃ increment. In the temperature of 38℃, the fluorescence intensity of the detection reaction attained the highest value. Then, the temperature was optimized in 38℃ (Figure 4B).\u003c/p\u003e\n\u003ch2\u003eSensitivity and specificity analysis of one-pot RT-RPA CRISPR/Cas12a detection reaction.\u003c/h2\u003e\n\u003cp\u003eTo determine the sensitivity of the detection reaction, the limit of detection (LoD) was verified by serially diluting the recombinant plasmid by two-fold, from 3.125 copies/test to 100 copies/test respectively. As shown in Figure 5, the LoD of present reaction was achieved 91.7 copies per test at the probability of 95%.\u003c/p\u003e\n\u003cp\u003eAs for specificity, a total of 26 virus samples were collected and used for detection, including 22 DENV samples and 4 non-DENV samples. All the DENV-positive samples were accurately identified, and no cross-reaction were observed, indicated the 100% specificity of present detection system for DENV (Figure 6A). Moreover, a total of 8 interferes virus samples were collected for detection, including four RNA samples and four DNA samples of sendai virus (SeV), Herpes simplex virus (HSV), Parainfluenza A virus (IAV), and vesicular stomatitis virus (VSV). The recombinant DENV plasmid was served as the positive control (PC) and the nuclease-free water was used as the negative control (NC). As shown in Figure 6B, only the PC induced a remarkable fluorescence intensity, and no remarkable signal were collected from the interfere samples. All these data suggested that the present system was specificity for DENV and no-cross reaction were identified.\u003c/p\u003e\n\u003ch2\u003eValidation of the one-pot RT-RPA CRISPR/Cas12a DENV detection system.\u003c/h2\u003e\n\u003cp\u003eTo examine the effectiveness of present platform for universal DENV detection, the recombinant plasmids that containing the corresponding nuclear acid sequence of DENV-I, DENV-II, DENV-III, and DENV-IV were synthesized and used for detection. As shown in Figure 7A, all these four serotypes of DENV were successfully identified by using present CRISPR-based detection system.\u003c/p\u003e\n\u003ch2\u003eEstablish and validation of the RT-RPA CRISPR/Cas12a-LFD DENV detection system.\u003c/h2\u003e\n\u003cp\u003eMeanwhile, we also constructed the DENV RT-RPA-CRISPR/Cas12a-LFD platform and used for detecting the four recombinant plasmid of DENV. As shown in Figure 7B, all the four serotypes of DENV were identified by using corresponding RT-RPA-CRISPR/Cas12a-colloidal gold test strip platform respectively (Figure 7B). Meanwhile, the FAM-T7-Biotin Reporter that used in the reaction was optimized by serially adjusting by two-fold from 200 nM to 25 nM respectively. As shown in Figure 7C, the efficiency of the test strip showed no significant difference by using the ssDNA reporter in different dose. For saving costs, the dose of the ssDNA reporter was determined in 25 nM for the test strip assay.\u003c/p\u003e\n\u003cp\u003eTo validate the sensitivity of the present test strip assay, the cultured DENV-II virus as indicated above were used for detection. The template of DENV-II nuclear acid samples was diluted into 2000 copies/test, 1000 copies/test, 500 copies/test, 250 copies/test, and 125 copies/test respectively. Four test replications were performed as each dose. As shown in Figure 7D, all the four replications were identified by using the template in the dose of 2000 copies/test, 1000 copies/test and 500 copies/test, while three for 250 copies/test and two for 125 copies/test respectively. All these results demonstrated that the present RT-RPA CRISPR/Cas12a-LFD was well-functioned for universal DENV detection.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDENV is a deadly virus that deeply threaten the human health and life quantity, over 4 billion people are living at a risk of dengue infection (23). However, the available therapy or vaccine are not approved at present. Therefore, an early detection method for DENV is essential to prevent its pandemic and optimize the coping strategies.\u003c/p\u003e\n\u003cp\u003eWith the rapid development of molecule detection techniques, the PCR-based method is widely applied as a routine assay for DENV diagnosis and offered high sensitivity (24-26). However, this method was limited by serval shortcomings, such as the requirement for expensive thermal cycling instrumentation, technical expertise, and prolonged testing times. Compared with PCR-based method, several reverse transcription recombinase polymerase amplifications (RT-RPA)-assist methods have been used for DENV detection and does not need thermal cycling instrumentation, which can performed as at constant temperature with limited testing time (27-29).However, the non-specific product during the amplification cannot totally avoided, resulting the high rate of false-positive results (30, 31). For this issue, Feng Zhang \u0026lsquo;s group have firstly established a SHERLOCK platform to detect DENV, offering high sensitivity to 2 aM. (13). However, this platform included two separated steps can only identify strain 1 or 3 of DENV, not available for the strain 2 or strain 4 of DENV and avoiding cross-contamination. Moreover, Vikrant Nain and colleagues have employed DENV-specific CRISPR-Cas13a gRNAs for the conserved and variable genomic regions among four DENV serotypes using bioinformatics tools, which can be used in diagnosing the dengue virus and its serotypes for \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003e(18). However, the performance of these gRNAs needs to be further validated. Huangxian Ju et.al have developed an electrochemical method based on CRISPR/Cas13a-assisted catalytic hairpin assembly (CHA) system for DENV detection, achieving the detection limit of 0.78 fM for detecting DENV type 1(32). However, this method requires high-cost electrochemical biosensor, professional technicians and cannot discriminate other type of DENV. In addition, Qianfeng Xia and colleagues have developed a bienzyme method for DENV detection based on Cas13a and Cas12a. (33). However, this platform can only detect DENV type 1 and not available for other types.\u003c/p\u003e\n\u003cp\u003eIn China, all the four serotypes and various genotypes of DENV strains have been identified and the incidence of DENV infection is increasingly serious in recent years (34, 35). Due to the multiple serotypes and genotypes of the DENV, most of the detection platform can only detected the dominant serotypes on specific regions and the universal DENV detection platform remains unknown by technique limitations.\u003c/p\u003e\n\u003cp\u003eIn the current study, we firstly developed a universal DENV detection system via combining RT-RPA amplification and CRISPR/Cas12a assay in one-pot. According to the multiple sequence alignment of four DENV serotypes, a pair of degenerate primers was selected and used for amplifying the target sequence that including all variations of these four DENV serotypes. Then, a Cas12a crRNA was used to recognize the target sequence. Importantly, the one-pot DENV universal detection system was well-functioned and successfully identified four recombinant plasmids that containing the conserved region of DENV-I to DENV-IV respectively. Moreover, the corresponding RT-RPA CRISPR/Cas12a-LFD platform was also established and showed a well performance by detecting the recombinant plasmid. As the LFD was cheap and convenient, our present DENV detection system has the potential value to extend for resources-limited regions, especially for developing countries and contributed to developing DENV Point-of-care Testing (POCT) and \u003cem\u003ein vitro\u003c/em\u003e diagnostic (IVD) products. Moreover, the whole assay can be finished in 40 min without the requirement for sophisticated equipment and cross operations. Moreover, there was no cross-reaction with other RNA viruses. Therefore, the RT-RPA CRISPR/Cas12a platform was extremely specificity for DENV, avoiding the likelihood of misdiagnosis. In addition, there was no need to uncapping procedures during the detection. Therefore, the current one-pot RT-RPA CRISPR/Cas12a assay yielded outstanding advantages for universal DENV detection, achieving rapid, accurate, simple and contamination-free properties.\u003c/p\u003e\n\u003cp\u003eFurthermore, all the component that can be pre-prepared by lyophilized powder and the whole assay can be performed on a simple thermal heater, even at room temperature. Therefore, the RPA CRISPR/Cas12a platform has the potential value to extend in resource-poor settings. Together, our present findings not only offered a promising available approach for universal DENV detection but also provided novel insight for developing the methods for identification other mankind viruses.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we introduced the RT-RPA assay and CRISPR/Cas12a system in one-pot for universal DENV detection and established the corresponding RT-RPA CRISPR/Cas12a-LFD system. The RT-RPA CRISPR/Cas12a method presented high specificity and sensitivity for DENV without false-positive results, achieving rapid, accurate, simple and contamination-free properties.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eY.Z., X.L. and M.R. supervised and secured funding for the project; Y.Z., X.L. and M.R. conceived the study, designed experiments, analyzed data, and revised the manuscript; Y.Z., Y.X. and D.H. designed experiments, performed experiments, analyzed data, interpreted data and drafted the manuscript. L.F., S.C., J.Z., Y.W., Y.J., B.L., J.B., Y.D., J.F. and S.C. performed experiments and collected data.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Key Research \u0026amp; Development Program of China (2023YFC2307302), the National Natural Science Foundation of China (32400727), the program of Shanghai outstanding academic leader in public health subject (GWVI-11.2-XD29), the experimental animal program sponsored by the Science and Technology Commission of Shanghai Municipality (23141902300), and Natural Science Foundation of Shanghai (24ZR1481100).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHarapan H, Michie A, Sasmono RT, Imrie A. 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Mol Cell Probes. 2019;46:101413.\u003c/li\u003e\n\u003cli\u003eLeon F, Pinchon E, Mayran C, Dayn\u0026egrave;s A, Morvan F, Mol\u0026egrave;s JP, et al. Magnetic Field-Enhanced Agglutination Readout Combined With Isothermal Reverse Transcription Recombinase Polymerase Amplification for Rapid and Sensitive Molecular Detection of Dengue Virus. Front Chem. 2021;9:817246.\u003c/li\u003e\n\u003cli\u003eAbd El Wahed A, Patel P, Faye O, Thaloengsok S, Heidenreich D, Matangkasombut P, et al. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection. PLoS One. 2015;10(6):e0129682.\u003c/li\u003e\n\u003cli\u003eJang M, Kim S. Inhibition of Non-specific Amplification in Loop-Mediated Isothermal Amplification via Tetramethylammonium Chloride. Biochip J. 2022;16(3):326-33.\u003c/li\u003e\n\u003cli\u003eGao X, Sun B, Guan Y. Pullulan reduces the non-specific amplification of loop-mediated isothermal amplification (LAMP). 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Reviews in medical virology. 2022;32(1):e2245.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"DENV, RPA, CRISPR/Cas12a, one-pot, LFD, universal detection ","lastPublishedDoi":"10.21203/rs.3.rs-5729352/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5729352/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Dengue Virus (DENV) is a severe life-threatening virus to human, which is the major cause for dengue fever. However, the efficiency of traditional detection methods unable to meet the additional requirements in clinic practice, including the virus isolation method, ELISA, RT-PCR and qRT-PCR and so on. Therefore, a rapid, simple, and accurate diagnostic for DENV is highly desired. In the current study, we developed a novel method for universal DENV detection via introducing recombinase polymerase amplification (RPA) assay and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and associated (Cas) protein 12a (CRISPR/Cas12a) system in one-pot, achieving the extremely sensitivity and specificity for DENV. The whole detection assay can be finished within 40 min without the requirement for sophisticated equipment. The limit of detection (LoD) was 91.7 copies per test. All the recombinant plasmid of these four serotypes of DENV (I to IV) were successfully identified by using present one-pot DENV universal RT-RPA CRISPR/Cas12a detection system. As for specificity, a total of 22 DENV positive samples were successfully identified, and no-cross reactions were observed in 4 other interferes nuclear acid samples. Moreover, we also established the universal DENV RT-RPA-CRISPR/Cas12a- lateral flow dipstick (LFD) platform and all the four serotypes of DENV (I to IV) were successfully identified, reaching the sensitivity of about 250 copies/test. Together, our present method not only provided an alternative approach for universal DENV detection but also gained a novel insight for other virus identification.","manuscriptTitle":"A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assay","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-06 05:00:52","doi":"10.21203/rs.3.rs-5729352/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-16T23:35:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-03T14:12:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-03T14:10:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2024-12-29T09:37:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5766ea33-cc40-4b10-bcd2-3cd5d223ca8e","owner":[],"postedDate":"January 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-31T16:06:21+00:00","versionOfRecord":{"articleIdentity":"rs-5729352","link":"https://doi.org/10.1186/s12866-025-03882-z","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2025-03-25 15:57:50","publishedOnDateReadable":"March 25th, 2025"},"versionCreatedAt":"2025-01-06 05:00:52","video":"","vorDoi":"10.1186/s12866-025-03882-z","vorDoiUrl":"https://doi.org/10.1186/s12866-025-03882-z","workflowStages":[]},"version":"v1","identity":"rs-5729352","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5729352","identity":"rs-5729352","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}