Concanavalin A-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay for rapid detection of HPV16

Concanavalin A-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay for rapid detection of HPV16 | 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 Concanavalin A-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay for rapid detection of HPV16 You Nie, Xiaohui Li, Wen Yang, Sihan Fei, Yingfan Wang, Yazhuo Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6278232/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 May, 2025 Read the published version in Microchimica Acta → Version 1 posted 11 You are reading this latest preprint version Abstract Human papillomavirus (HPV) infection is a major threat to women’s health worldwide. High-risk subtypes, particularly HPV16, require rigorous screening and long-term surveillance to control cervical cancer. However, traditional HPV testing is hampered by the need for nucleic acid extraction, reliance on specialized technicians, and fluorescence detection equipment, limiting its suitability for rapid on-site testing. In this study, we developed a Concanavalin A-assisted extraction-free one-pot recombinase polymerase amplification (RPA) CRISPR/Cas12a assay (ConRCA) for HPV16. Concanavalin A-coated magnetic beads were used for target enrichment and nucleic-acid-extraction-free processing. Suboptimal protospacer-adjacent motifs were used to achieve a one-pot RPA–CRISPR/Cas12a assay. The ConRCA assay can be completed in approximately 25 min under isothermal conditions and can detect at least 1.2 copies/µL of HPV16 genomic DNA using a fluorescence reader or test strip. The feasibility of this detection method was evaluated with 31 unextracted clinical samples. Compared with qPCR, the overall sensitivity was 95% (19/20), and the specificity was 100% (11/11). Our results indicate that the ConRCA assay has great potential utility as a point-of-care testing for the rapid identification of HPV. Concanavalin A Extraction-free assay CRISPR/Cas12a One-pot detection HPV Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Human papillomavirus (HPV) infection is a global health concern. HPV is an enveloped double-stranded DNA virus that exclusively infects humans, and is responsible for 95% of cervical cancers. It is also the causative agent in approximately 5% of human cancers, including anal, vaginal, vulvar, and penile cancers [ 1 , 2 ] . HPV16 is the most important subtype of the 12 high-risk carcinogenic subtypes, and it is associated with 62.4% of cervical cancers [ 3 ] . Therefore, screening for high-risk subtypes, particularly HPV16, and long-term monitoring are crucial measures for preventing the occurrence of cervical cancer. Currently, polymerase chain reaction (PCR) is a widely used HPV detection technique, known for its high sensitivity and reproducibility [ 4 ] . However, due to regional conditions, limited technical personnel, and the restriction of detection equipment to specialized laboratories, the coverage and follow-up rates of HPV screening are insufficient [ 5 , 6 ] . Therefore, the development and implementation of point-of-care testing technologies with broader applicability, to promote pre-hospital HPV screening, are important measures for improving the quality and coverage of cervical cancer screening. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system was initially discovered as an adaptive immune mechanism in bacteria. Today, it has demonstrated strong competitiveness in the fields of gene editing and pathogen diagnosis. In particular, some Cas proteins with special functions in this system, such as Cas12 and Cas13, can recognize target sequences and initiate targeted cleavage activity under the guidance of guide RNA (gRNA). These proteins are not only capable of cleaving target DNA or RNA, but also have ‘trans-cleavage’ abilities, meaning that they can cleave other nontargeted DNA or RNA molecules. By combining reporter nucleic acid molecules labeled with fluorescent groups and utilizing the trans-cleavage properties of Cas proteins, the detection of target genes can be achieved [ 7 – 9 ] . The recombinase–polymerase amplification (RPA)–CRISPR/Cas12a method combines the high specificity of CRISPR/Cas12a with the high sensitivity of RPA, allowing the efficient amplification, recognition and cleavage of nucleic acid target sequences in a short time, and significantly enhancing the detection sensitivity. This method has been widely used to analyze the nucleic acids of pathogenic microorganisms, such as Staphylococcus aureus , Salmonella , and SARS-CoV-2 [ 10 ] . However, traditional RPA–CRISPR/Cas12a detection techniques typically require two separate reaction steps, nucleic acid amplification and CRISPR-mediated signal detection. This stepwise operation is relatively cumbersome, increasing the complexity and duration of the experiment. Moreover, the process of transferring amplified products increases the likelihood aerosol cross-contamination and false-positive results. These issues make the technique less than ideal for practical applications, especially in scenarios requiring high-throughput and rapid detection [ 11 – 13 ] . So far, many studies have aimed to integrate the two parts within a single tube using physical separation. This strategy involves adding nucleic acid amplification and CRISPR detection components to the bottom and the wall of the reaction tube, respectively. After the amplification process is completed, mixing is achieved with centrifugation [ 14 , 15 ] . Tan et al [ 16 ] also reported a one-pot method in which the RPA and CRISPR/Cas12a reactions were separated by paraffin, creating an independent platform upon which the RPA reaction generates sufficient target product before its combination with the CRISPR/Cas12a system. A light-controlled one-pot method for activating Cas12a was also developed to enhance the accuracy and sensitivity of the technique [ 17 ] . These methods effectively address the issue of aerosol contamination, but they often require additional centrifugation or activation steps, which increase the complexity and cost of the detection process. Overall, achieving the efficient integration of nucleic acid amplification with the CRISPR/Cas system within a single reaction tube still presents technical challenges, limiting its potential application to rapid on-site testing. Sample preprocessing is also a significant factor limiting on-site application. Two commonly used nucleic acid extraction methods, column- and magnetic-bead-based methods, can provide high-purity nucleic acid molecules, but they require repeated pipetting and centrifugation, which are inconvenient operations. Moreover, the need to repeatedly open and close the tubes during nucleic acid extraction can lead to cross-contamination. In recent years, to overcome the drawbacks of traditional nucleic acid extraction methods, various simplified nucleic acid extraction schemes have been proposed. These methods often involve direct lysis techniques that depend on chemical methods, enzymatic methods, or high temperatures. However, for samples with low-concentration targets, these methods can further reduce the probability of isolating the target nucleic acids, thereby reducing the sensitivity of the detection [ 11 ] . These direct lysis methods also involve a large number of complex components, which may inhibit subsequent amplification and signal readout. Therefore, obtaining target nucleic acids with high purity in a rapid, simple, and efficient manner is a challenge currently faced in sample preprocessing. Lectins are a group of heterogeneous proteins widely found in various plants, invertebrates, and higher animals. They possess specific recognition regions (carbohydrate recognition domains) that can identify and reversibly bind to specific carbohydrates [ 18 ] . Concanavalin A (ConA) is a lectin extracted from leguminous plants, which was among the earliest to be characterized and extensively studied. Research has shown that, in addition to glucose and mannose, which significantly inhibit the hemagglutination activity of ConA, galactose, lactose, trehalose, rhamnose, N-acetyllactosamine, N-acetylglucosamine, sucrose, and maltose also exert inhibitory effects on the hemagglutination activity of ConA [ 19 ] . This indicates that ConA has a high affinity for these sugar molecules. Therefore, it can be used in the separation and purification of glycosylated substances and viruses, and in the field of biological diagnostics [ 18 , 20 ] . Several studies have reported the use of ConA in pathogen detection. Kim [ 21 ] developed diagnostic kits for norovirus in which ConA was used to concentrate norovirus. Zhang [ 22 ] demonstrated that ConA-modified biofunctional nanoparticles have a minimum limit of detection (LOD) 50 CFU/mL for Gram-positive bacteria. ConA binds to the glycosylation sites of human immunodeficiency virus (HIV) gp120 and overcomes strain limitations [ 23 ] . This offers a new direction for the development of detection tools for HPV. Another study demonstrated the glycosylation modification of the HPV L1 protein, which ensured the correct folding and structural stability of the protein [ 24 ] . A recent study reported that after HPV infection, the level of O-GlcNAc glycosylation in cells increases, and that the sugar structure involved in this glycosylation modification is mainly O-linked β-N-acetylglucosamine (O-GlcNAc). This modification involves the attachment of a single N-acetylglucosamine (GlcNAc) residue to a serine (Ser) or threonine (Thr) residue of proteins via a β-glycosidic bond [ 25 , 26 ] . These finds provides inspiration for the detection of HPV based on ConA. In this study, we developed a ConA-assisted extraction-free one-pot RPA–CRISPR/Cas12a assay (ConRCA) for the point-of-care detection of HPV. This method utilizes ConA-coated magnetic beads (ConA MBs) for viral pre-enrichment and combines RPA and the CRISPR/Cas12a recognition amplification system in a single reaction tube. This approach increases the sample concentration and significantly reduces the aerosol contamination caused by repeatedly opening and closing the tube, thus reducing the operational complexity. The results are visualized with fluorescence or lateral flow strips, allowing the visual detection of HPV16 within 25 min. The assay is characterized by its high sensitivity, high specificity, and strong versatility, and provides a reliable and effective technique for the rapid diagnosis and long-term monitoring of HPV patients. Materials and methods Materials The hiScribe T7 quick high yield RNA synthesis kit, Lba Cas12a, 10× NEBuffers 2.1 and RNase inhibitor was obtained from New England Biolabs (Beijing, China). The HPV plasmid, primers, and ssDNA reporters (single-strand DNA reporters, FAM-TTATT-BHQ1 and FAM-TTATTATT-Biotin) were synthesized by Sangon Biotech (Shanghai, China). The DNA isothermal rapid amplification kit was purchased from Amp Future Bio-Tech Co (Changzhou, China). Lateral flow readout strip were purchased from Tolo Biotech. (Shanghai, China). High risk human papillomavirus (HPV) nucleic acid assay kit (qPCR assay) from Liferiver (Shanghai, China). The EasyPure ® RNA kit purchased TransGen Biotech (Beijing, China). ConA MBs were obtained from Biyun Tian Biotechnology Co., Ltd (Shanghai, China). Pathogenic microorganisms DNA/RNA extraction kit was obtained from Beaver (Suzhou, China). Nucleic acid releasing agent was obtained from Amp Future Bio-Tech Co (Changzhou, China). Preparation of single guide RNA The DNA template for in vitro transcription is two reverse complementary paired oligos containing a T7 promoter sequence and spacer sequence. The two DNA templates were annealed at 85°C to 25°C. The annealed product was incubated with T7 RNA polymerase for in vitro transcription at 37°C for 3h. The in vitro transcription reaction was treated with DNase I (Promega) for 10 min at 37°C, and then purified using the The EasyPure ® RNA Kit. The purified sgRNA is stored at -80°C. Design and screening of RPA primers and sgRNAs The six specific RPA amplification primers and eight specific sgRNAs were designed (Table S1 and Table S2 ) using SnapGene 6.0.2 software. Three upstream primers and two downstream primers in two-by-two combinations totaled six sets. The eight sgRNAs contained four classical protospacer-adjacent motif (PAM) (TTTN) site sgRNAs and four suboptimal PAM (VTTV) site sgRNAs. The RPA products were screened for RPA primers by 1.5% agarose gel electrophoresis, and then the screened RPA primer sets were using CRSIPR/Cas12a fluorescence assay to select sgRNAs. RPA and qPCR assay Standard RPA reactions were conducted according to the DNA isothermal rapid amplification kit’s protocol. The RPA amplification mix consisted of 29.4 µL of A Buffer, 10 µM each of forward and reverse primers, 2.5 µL of A Buffer, 2 µL of DNA template, and ddH 2 O to obtain a final volume of 50 µL. The reactions were performed at 37°C for 30 min. The HPV qPCR assay were performed in 40 µL reaction volumes containing 36 µL of qPCR mixture and 4 µL lysed sample. The reaction by incubating at 94°C for 2 min, followed by 40 cycles of 93°C for 10 s, 62°C for 31 s. ConRCA systems One-pot reactions systems were performed in 40 µL reaction volumes containing 25 ~ 100 nM LbCas12a, ssDNA reporter was 400 ~ 1000 nM (FAM-TTATT-BHQ1) or 25 ~ 50 nM (FAM-TTATTATT-Biotin), 25 ~ 200 nM sgRNA, 0.25× ~2× NEBuffer 2.1, 18 µL RPA mixture. The 2.5 µL B Buffer and 8 µL tested sample were added to each one-pot reaction systems. The readout through SpectraMax i7x or lateral flow strip at 37°C for 15–60 min. ConA enrichment and HPV DNA extraction To simulate real detection scenarios, a sample pool was created by combining 1 mL each of 10 HPV16-positive samples, generating a 10 mL bulk sample. Enrichment analyses were conducted with 200 µL aliquots, to which another 200 µL of 2× binding buffer (40 mM HEPES [pH7.4], 2 mM MgCl 2 , 2 mM MnCl 2 , 2 mM CaCl 2 ) was added. Each 400 µL sample was spiked with ConA MBs at 0.5, 1, 3, 5, 10, or 15 µL of the total sample volume and incubated at room temperature for 1, 5, 10, 15, 20, 25, or 30 min. The ConA MBs were magnetically pelleted and the supernatant was removed. The ConA MBs were resuspended in 50 µL of nucleic-acid-releasing agent before incubation for 5 min at 100°C. The appropriate volumes of these positive pools were then combined with negative clinical samples diluted 1:10 and 1:100 for the recovery experiment. The original samples and diluted samples were each divided into two equal portions. The DNA in one was directly extracted with a commercial nucleic acid extraction kit and the products were quantified with quantitative PCR (qPCR). The other portion was enriched with ConA MBs before all of the mixture was subjected to nucleic acid extraction and the products were quantified with qPCR. Nucleic acid extraction was performed according to the manufacturer’s instructions. Collection of HPV16 clinical samples HPV samples were collected in March–September 2024 at the PLA General Hospital Fourth Medical Center, Beijing, China. The sample swabs were placed in a centrifuge tube containing 1 mL of saline. The processing of clinical samples was performed in accordance with the Materials and Methods section 2.6. Results and Discussion Principle of ConRCA The design and reaction mechanism of ConRCA are shown in Fig. 1 . This strategy utilizes ConA MBs to rapidly enrich the target and release the nucleic acids within 10 min, avoiding the cumbersome sample pretreatment process required in conventional methods. The release of viral DNA was accomplished with a nucleic-acid-releasing agent. This one-step extraction method achieves effective enrichment of low-concentration targets and the rapid release of nucleic acids, eliminating the need for time- and labor-intensive column purification and bypassing centrifugation. By designing two types of sgRNAs with canonical or suboptimal PAM sites, the trans-cleavage efficiency of Cas12a/sgRNA was verified. When a fluorescence reader or lateral flow strip was used, ConRCA detected as little as 1.2 copies/µL of HPV DNA within 15 min. Several reports have indicated that the collateral cleavage activity of Cas is closely related to sgRNAs with different PAM sites. Given that the ConRCA developed in this study necessitates the synchronization of RPA and Cas12a cleavage, the method is contingent on the slower enzymatic kinetics of Cas12a. [ 27 , 28 ] First, Cas12a is activated using the rapidly accumulating amplicons in the pre-reaction stage. The single-stranded DNA (ssDNA) probe is then sheared by a large number of activated Cas12a. Finally, the rapidly accumulating probe signals are read. Notably, the method does not require a complex way to physically isolate the RPA and CRISPR/Cas reaction systems. The simplicity, speed, and stability of the method makes it suitable for on-site nucleic acid testing, and obviates the need for specialized laboratories and healthcare professionals. Optimization of ConA-assisted enrichment conditions The parameters for target enrichment, including the input volume of ConA MBs (10 mg/mL) and the incubation time, were optimized to minimize the resultant cycle threshold (Ct) values. First, parallel aliquots (200 µL) of 10 positive clinical samples mixing were analyzed with volumes of ConA MB of 0.5–15 µL. The optimal ConA MB input volume was determined to be 10 µL, because 0.5–5 µL and 15 µL produced higher relative Ct values, indicating lower DNA concentrations after extraction, whereas an input volume of 10 µL produced the lowest Ct value (Fig. 2 A). This ConA MB input volume was used to determine the optimal incubation time in the range of 1–30 min. The ideal incubation time was determined to be 5 min. The highest Ct value was observed at 1 min, and the values tended to converge after 5 min (Fig. 2 B). To assess the efficacy of ConA-mediated target enrichment, we conducted tests on two 200 µL samples with identical dilution factors, as detailed in the Materials and methods section 2.6. Specifically, one sample was subjected to commercial nucleic acid extraction followed by qPCR to determine the Ct value. The other sample was enriched with ConA, and the supernatant was discarded after magnetic selection. The magnetic beads were then resuspended in 200 µL of nuclease-free water. This resuspended mixture was used as the new sample for commercial nucleic acid extraction and subsequent qPCR analysis. This procedure was designed to negate any variability introduced by the nucleic acid extraction process, thus providing a more-precise evaluation of the enrichment efficiency. Two-way ANOVA with replication revealed no significant difference between the means (α = 0.05, P = 0.2238), indicating that the Ct values obtained during DNA amplification with both methods were statistically indistinguishable across all dilutions (Fig. 2 C, D). This finding suggests that the ConA-assisted assay gives essentially the same results as the conventional nucleic acid extraction method at the concentrations tested. Primer and sgRNA screening Six pairs of specific RPA primers were designed (Fig. 3 A, Fig. S1 , Supplementary Table 1). Including three forward primers and two reverse primers, which were paired in all possible combinations, resulting in a total of six different primer pairs. Agarose gel electrophoresis of the RPA products showed that when the concentration of the amplification template was 1.2 × 10³ copies/µL, the primer combinations F1R1, F2R1, F3R1, and F3R2 all successfully amplified the target fragment (Fig. 3 B). The target band (230 bp) amplified by the primer combination F3R1 was brightest. Therefore, the optimal primer combination F3R1 was selected for the subsequent one-pot reaction systems examined in this study. Based on the conserved sequences in the F3R1 primers, eight sgRNAs with different PAM sites (including the classic TTTN and suboptimal VTTV motifs) were designed (Fig. 3 A, Table S2 ). In the one-pot reaction system, sgRNA and Cas12a acted as the guide RNA and nuclease, respectively, to form a ribonucleoprotein complex (RNP). We then investigated the cleavage effects of the RNPs formed by two sgRNAs with different PAM sites and Cas12a. In this study, two-step and one-pot assays for fluorescence detection were used to screen the sgRNAs. The result of the two-step assay showed that canonical sgRNA1 and sgRNA4 both produced fluorescent signals, and that canonical sgRNA1 had the highest relative fluorescence value (Fig. 3 C). The results of the one-pot assay showed that all four suboptimal sgRNAs produced fluorescent signals, and that suboptimal sgRNA1 had the highest relative fluorescence value (Fig. 3 D). To compare the effects of the optimal canonical sgRNA and suboptimal sgRNA on the cleavage efficiency of Cas12a, we used both two-step and one-pot assays with the selected canonical sgRNA1 and suboptimal sgRNA1. The results indicated that in the two-step assay, canonical sgRNA1 yielded a higher fluorescent detection value than suboptimal sgRNA1 (Fig. 3 E). In contrast, in the one-pot assay, suboptimal sgRNA1 demonstrated an earlier onset of fluorescence and a higher fluorescence value than canonical sgRNA1 (Fig. 3 F). These findings are consistent with previous studies that have explored the influence of different PAM sites on Cas12a activity. [ 28 ] It has been demonstrated in previous studies that the design of sgRNA based on different PAM sites directly affects the cleavage efficiency of Cas12a. It is noteworthy that within one-pot reaction systems, Cas12a guided by a suboptimal sgRNA shows better cleavage efficiency than Cas12a guided by canonical sgRNA [ 29 – 31 ] . These findings suggest that the use of suboptimal sgRNA in one-pot reaction systems allows the acquisition of results in as little as 15 min, without compromising the detection accuracy (Fig. 3 F). This approach has been shown to significantly reduce the detection time and simplify the procedure, thereby minimizing the risk of aerosol nucleic acid contamination. Consequently, suboptimal sgRNA1 was selected for subsequent experiments in this study. Optimization of CRISPR reaction parameters To enhance the cost-effectiveness and performance of the one-pot reaction system, we optimized the working concentrations of critical components within the reaction system. These components included Cas12a, the ssDNA probe, NEBuffer 2.1, and sgRNA. As shown in Fig. 4 A, the detected fluorescence values were significantly higher when Cas12a was at a concentration of 50 nM than at 25 nM or 100 nM. The lower fluorescence value at 100 nM may be attributable to the glycerol concentration in the protein preservation solution. There are many free molecules in one-pot reaction systems, and higher concentrations of glycerol will affect the free state of each molecule, which in turn will affect the reaction efficiency. Therefore, the optimal working concentration of Cas12a was selected as 50 nM in this study. We next tested the effect of different concentrations of the ssDNA fluorescent probe. Previous studies have reported Cas12a to ssDNA ratios of 1:7 or 1:10 [ 32 , 33 ] . As shown in Fig. 4 B, the detected fluorescence values gradually increased as the working concentration of the ssDNA fluorescent probe increased. When the concentration of the ssDNA probe increased from 800 nM to 1 µM, there was a noticeable acceleration in the rate of fluorescence increase. Consequently, 1 µM was selected as the optimal working concentration of the ssDNA probe. The ratio of Cas12a to ssDNA was 1:4. We next optimized the amount of NEBuffer 2.1 added to the reaction system. As shown in Fig. 4 C, 2 × NEBuffer 2.1 produced the lowest detected fluorescence value, and 0.25 × NEBuffer 2.1 produced the highest detected fluorescence value. The reason may be that the high concentration of salt ions had some inhibitory effect on RPA. Therefore, we selected the optimal working concentration as 0.25 × NEBuffer 2.1. Finally, we optimized the sgRNA concentration, as shown in Fig. 4 D. The highest fluorescence value was detected when the working concentration of sgRNA was 100 nM, which selected as the optimal working concentration. In summary, the optimized working concentration ratio was Cas12a: sgRNA: ssDNA = 1:2:4. Sensitivity and specificity of ConRCA A gradient dilution of the HPV16 DNA plasmid (1.2 × 10 5 , 1.2 × 10 4 , 1.2 × 10 3 , 1.2 × 10 2 , 1.2 × 10 1 , 1.2 × 10 0 , 1.2 × 10 −1 copies/µL) was used for the qPCR assay. As shown in Fig. 5 A, the LOD of the qPCR was 1.2 × 10 0 copies/µL, whereas 1.2 × 10 −1 copies/µL was not detected. The sensitivity of the fluorescence method and lateral flow strip was evaluated with ConRCA. As demonstrated in Fig. 5 B–F, the sensitivity of the fluorescence and lateral flow strip methods was 1.2 × 10 0 copies/µL, consistent with the qPCR results. Notably, the ConRCA reaction took only 15 min (Fig. 5 D). Compared with the detection times documented in previous studies (85 min, 60 min, and 45 min), the operation was more expeditious and efficient in this study. This demonstrates the potential for the development of on-site detection or at-home self-testing technologies [ 33 – 35 ] . To evaluate the specificity of ConRCA, we tested six nucleic acid templates: HPV16, human immunodeficiency virus (HIV), hepatitis B virus (HBV), HPV18, HPV6, and HPV11. The test was performed with the ConRCA fluorescence readout, as shown in Fig. 6 A, B. This method specifically detected HPV16, with no obvious cross-reaction with the other pathogens tested. The test was then performed with the ConRCA lateral flow strips readout, as shown in Fig. 6 C, D. The test strips specifically showed HPV16-positive bands after a 15 min reaction time, with no obvious cross-reactivity with the other pathogens tested, and the results were consistent with those of the fluorescence method. Examination of the clinical samples The vaginal secretions from HPV-infected patients are characterized by their diversity and complexity, posing a challenge to existing methods of rapid HPV nucleic acid testing [ 36 ] . In this study, we developed a simple sample pretreatment technique using ConA-MBs, as shown in Fig. 1 , which achieved efficient nucleic acid release within 10 min. To evaluate the performance of ConRCA in detecting HPV16 in clinical samples, 31 clinical samples were collected, including 21 HPV16-positive samples with different viral loads (sample 1–21) and 10 HPV-negative samples (sample 22–31). The qPCR method was first used to analyze these samples. As shown in Fig. 7 A, the positive detection rate for the qPCR method was 95% (20/21). The result for sample 6 was negative, which could be attributed to the degradation of the nucleic acids during storage and transportation. ConRCA was then used to evaluate the same samples with a fluorescence reader or lateral flow strip. As shown in Fig. 7 A and Fig. S2 , sample 6 was also negative with both the fluorescence and the strip test, consistent with the results of qPCR. Notably, the result for sample 15 using the strip test was negative (Fig. 7 A). The Ct value of 38.12 for sample 15 and the low relative fluorescence value detected with the ConRCA fluorescence strategy suggest that the sensitivity of the test strip may be slightly lower than that of the ConRCA fluorescence strategy and qPCR in clinical samples, which is in general agreement with the results of a previous study [ 37 ] . Based on the results of the three methods described above, the nucleic acids in sample 6 had been degraded, leading to a negative result. Using the qPCR results as the gold standard, the sensitivity of the ConRCA fluorescence strategy was calculated to be 100% (20/20) and the specificity was also 100% (11/11) when ConRCA was used to test clinical samples. The sensitivity of the lateral flow strip method was 95% (19/20) and the specificity was 100% (11/11). The overall sensitivity was 95% (19/20) and the specificity was 100% (11/11) (Fig. 7 B). From the receiver operating characteristic (ROC) curve obtained from the statistical analysis shown in Fig. 7 C, the area under the curve (AUC) for ConRCA was 0.9722 (95% confidence interval: 0.9243–1.0000). These findings demonstrate the efficacy of the established method in accurately and reliably detecting HPV16 in actual samples. Conclusion In summary, we have developed a ConA-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay, designated ConRCA, for the rapid on-site detection of HPV16. The results can be evaluated with a fluorescence reader or lateral flow strip within 25 min. The LOD for HPV16 DNA is 1.2 copies/µL. The results for 31 clinical samples demonstrated that ConRCA has a sensitivity of 95.00% and a specificity of 100.00%, which is consistent with the qPCR technology. ConRCA allows rapid and sensitive testing with simple operation, offering significant advantages for on-site diagnosis. Furthermore, the development of multichannel microfluidic chips that integrate primers and sgRNAs specific to various HPV types has potential utility in high-throughput HPV screening and at-home self-examination. Declarations Author contributions You Nie: Writing – original draft, Visualization, Validation, Methodology, Investigation, Conceptualization. Xiaohui Li: Writing – original draft, Validation, Methodology, Investigation, Conceptualization. Wen Yang: Writing – original draft, Visualization, Validation, Methodology, Investigation. Sihan Fei: Collection of clinical samples. Yingfan Wang: Resources, Data curation. Yazhuo Li: Software, Data curation. Ke Zhang: Software, Data curation. Jiarui Kang: Software, Data curation. Yang Cheng: Formal analysis, Data curation. Hongwei Wang: Writing – review & editing, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. Dandan Liu: Writing – review & editing, Validation, Supervision, Methodology, Investigation, Conceptualization. Acknowledgments Not applicable. Funding This work was supported by the Youth Independent Innovation Science Foundation of the General Hospital of the Chinese People's Liberation Army (grant number 22QNFC098) and the Military Family Planning Program of China (grant number 24JSZ12). Ethics declaration and consent to participate This study was approved by the ethics committee of the PLA General Hospital Fourth Medical Center in accordance to the Helsinki Declaration (S2021-564-01). All the procedures were performed in accordance with the Declaration of Helsinki, and the patient’s confidentiality was maintained throughout the investigation. All individuals provided their written informed consent to participate in this study. All methods were carried out in accordance with relevant guidelines and regulations. Consent for publication Not applicable. 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Nat Biomed Eng 6(3):286–297 Yamano T, Zetsche B, Ishitani R et al (2017) Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1. Mol Cell 67(4):633–645e3 Nalefski EA, Kooistra RM, Parikh I et al (2024) Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets. Nucleic Acids Res 52(8):4502–4522 Patchsung M, Jantarug K, Pattama A et al (2020) Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng 4(12):1140–1149 Zhang HX, Zhang C, Lu S et al (2023) Cas12a-based one-pot SNP detection with high accuracy. Cell Insight 2(2):100080 Gong S, Song K, Zhang S et al (2024) CRISPR-Cas12a-mediated dual-enzyme cascade amplification for sensitive colorimetric detection of HPV-16 target and ATP. Talanta 266(Pt 2):125050 Zeng Q, Zhou M, Hu Z et al (2023) Rapid and sensitive Cas12a-based one-step nucleic acid detection with ssDNA-modified crRNA. Anal Chim Acta 1276:341622 Zhao Y, Chen D, Xu Z et al (2023) Integrating CRISPR-Cas12a into a Microfluidic Dual-Droplet Device Enables Simultaneous Detection of HPV16 and HPV18. Anal Chem 95(6):3476–3485 Sormani J, Kenfack B, Wisniak A et al (2021) Exploring Factors Associated with Patients Who Prefer Clinician-Sampling to HPV Self-Sampling: A Study Conducted in a Low-Resource Setting. Int J Environ Res Public Health, 19(1) Hu J, Yu Y, Pan X et al (2024) Highly sensitive and specific detection of human papillomavirus type 16 using CRISPR/Cas12a assay coupled with an enhanced single nanoparticle dark-field microscopy imaging technique. Talanta 278:126449 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx RevisedSupplementaryMaterial.docx Graphicalabstract.tif Cite Share Download PDF Status: Published Journal Publication published 14 May, 2025 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 08 Apr, 2025 Reviews received at journal 07 Apr, 2025 Reviews received at journal 06 Apr, 2025 Reviews received at journal 04 Apr, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 30 Mar, 2025 Reviewers agreed at journal 30 Mar, 2025 Reviewers invited by journal 30 Mar, 2025 Editor assigned by journal 26 Mar, 2025 Submission checks completed at journal 25 Mar, 2025 First submitted to journal 21 Mar, 2025 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. 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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-6278232","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440188052,"identity":"03759c58-7b72-4135-b044-220a278cfe57","order_by":0,"name":"You Nie","email":"","orcid":"","institution":"Department of Pathology, Fourth Medical Centre of Chinese PLA (People’s Liberation Army) General Hospital","correspondingAuthor":false,"prefix":"","firstName":"You","middleName":"","lastName":"Nie","suffix":""},{"id":440188053,"identity":"c6d60a88-860e-4965-84f7-77ba92e9f148","order_by":1,"name":"Xiaohui Li","email":"","orcid":"","institution":"College of Veterinary Medicine, Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Li","suffix":""},{"id":440188054,"identity":"49729c40-c635-466f-8797-9fbb8991d02e","order_by":2,"name":"Wen Yang","email":"","orcid":"","institution":"Department of Gynaecology and Obstetrics, Seventh Medical Centre of Chinese PLA (People’s Liberation Army) General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Yang","suffix":""},{"id":440188055,"identity":"373d2815-7be6-455f-b5ba-fd9cecfa1a69","order_by":3,"name":"Sihan Fei","email":"","orcid":"","institution":"College of Veterinary Medicine, Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Sihan","middleName":"","lastName":"Fei","suffix":""},{"id":440188056,"identity":"6e5a09c6-10bf-42ec-afe6-684ea502f893","order_by":4,"name":"Yingfan Wang","email":"","orcid":"","institution":"Department of Gynaecology and Obstetrics, The Third Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yingfan","middleName":"","lastName":"Wang","suffix":""},{"id":440188057,"identity":"e5fb487f-4819-4e09-87d6-4678a28ddfd8","order_by":5,"name":"Yazhuo Li","email":"","orcid":"","institution":"Department of Pathology, Fourth Medical Centre of Chinese PLA (People’s Liberation Army) General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yazhuo","middleName":"","lastName":"Li","suffix":""},{"id":440188058,"identity":"ed3a7937-825f-48ae-ab13-961bcd81618a","order_by":6,"name":"Ke Zhang","email":"","orcid":"","institution":"Department of Pathology, Fourth Medical Centre of Chinese PLA (People’s Liberation Army) General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Zhang","suffix":""},{"id":440188059,"identity":"e30aa920-f51e-483b-8ac2-5d7ac773bc0a","order_by":7,"name":"Jiarui Kang","email":"","orcid":"","institution":"Department of Pathology, Fourth Medical Centre of Chinese PLA (People’s Liberation Army) General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiarui","middleName":"","lastName":"Kang","suffix":""},{"id":440188061,"identity":"4ef76b42-925e-4bc0-a792-2cfab8cdf411","order_by":8,"name":"Yang Cheng","email":"","orcid":"","institution":"Department of Pathology, Fourth Medical Centre of Chinese PLA (People’s Liberation Army) General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Cheng","suffix":""},{"id":440188063,"identity":"3147bd30-54ec-4c5f-9597-1eb357f9dcb6","order_by":9,"name":"Hongwei Wang","email":"","orcid":"","institution":"Department of Pathology, Fourth Medical Centre of Chinese PLA (People’s Liberation Army) General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongwei","middleName":"","lastName":"Wang","suffix":""},{"id":440188065,"identity":"9bdc4c88-7ed0-42b8-8dbc-fd90f50df57d","order_by":10,"name":"Dandan Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACfvbGxgcfKmrk+NkbiNQi2XP4sOGMM8eMJXsOEKnF4EZamjRnC3PihhsJxGo5kGMmzdjAxrjh5uONNxhqbKIJO+zAGWPrwh0yzJK304otGI6l5TYQ0sJ3sMfw9swzbGx8t3PMJBgbDhPWwnCYx0Cat42Zh+HmGSK1CBxjSwJpkRC4wUOkFskeZnAgG0j2AP2SQIxf+OUfgqOyvp/98MYbH2psiPALEjCQSCBFOUQLqTpGwSgYBaNgZAAApRhEksD7XjcAAAAASUVORK5CYII=","orcid":"","institution":"Department of Pathology, Fourth Medical Centre of Chinese PLA (People’s Liberation Army) General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-03-21 13:53:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6278232/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6278232/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-025-07198-7","type":"published","date":"2025-05-14T15:57:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80921442,"identity":"7b45d40b-8bd5-4271-ab4b-2c1e44d63df3","added_by":"auto","created_at":"2025-04-18 20:48:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10612826,"visible":true,"origin":"","legend":"\u003cp\u003ePrinciple of ConA-assisted one-pot RPA-CRISPR/Cas12a assay.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/8ba1b19dd70fbd86c9697cf5.png"},{"id":80921439,"identity":"4f88fb01-a04b-4edb-80f5-16c23c611f12","added_by":"auto","created_at":"2025-04-18 20:48:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3046240,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of enrichment conditions. (A) ConA MBs input volume. (B) Incubation time of ConA MBs. (C, D) Clinical samples were extracted by the commercial nucleic acid extraction kit or proposed ConA-assited assay. No statistical differences were observed between Ct values resulting from amplification of eluates produced by either method.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/4d753c4219ca67048854d93c.png"},{"id":80921444,"identity":"0024d112-f4be-4ac3-a7e3-7559ad6e7f6c","added_by":"auto","created_at":"2025-04-18 20:48:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9061504,"visible":true,"origin":"","legend":"\u003cp\u003ePrimer and sgRNA design and screening. (A) Schematic of primer and sgRNA design. (B) Screening of RPA primers by agarose gel electrophoresis. The red dashed box is the brightest band and NC refers to the negative control. (C) Two-pot assay screening of canonical sgRNAs. (D) One-pot assay screening of suboptimal sgRNAs. (E) Two-pot assay for different types of sgRNA. (F) One-pot assay for different types of sgRNA.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/2ccc8e1024b320398753b55f.png"},{"id":80921459,"identity":"c8c252db-e491-4c39-b661-1ee68edfcc22","added_by":"auto","created_at":"2025-04-18 20:48:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6511504,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of CRISPR reaction parameters. (A) Optimization of ssDNA reaction concentration. (B) Optimization of Cas12a reaction concentration. (C) Optimization of NEBuffer 2.1 reaction concentration. (D) Optimization of sgRNA reaction concentration. NC refers to the negative control.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/a8f06d3714aa1b5d944d9470.png"},{"id":80921888,"identity":"0d8475d9-3011-454d-b520-ffd5385e388e","added_by":"auto","created_at":"2025-04-18 21:04:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10587447,"visible":true,"origin":"","legend":"\u003cp\u003eThe sensitivity evaluation of ConRCA. (A) Detection of HPV16 with qPCR. (B) ConRCA fluorescence assay for HPV16. (C) ConRCA fluorescence assay for HPV16 at 1 h. (D) ConRCA fluorescence assay for HPV16 at 15 min. (E) ConRCA lateral flow strip readout for HPV16. (F) ConRCA lateral flow test strip to read HPV16 band intensity. NC refers to the negative control. C: control line; T: test line. “****” P \u0026lt; 0.0001, “***” P \u0026lt; 0.005，“*” P \u0026lt; 0.05; “ns” : not significant.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/11dcc09d35c63377025f3637.png"},{"id":80921462,"identity":"1ba4aa13-229c-4e34-8483-1eebfc3ec716","added_by":"auto","created_at":"2025-04-18 20:48:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3729108,"visible":true,"origin":"","legend":"\u003cp\u003eThe specificity evaluation of ConRCA. (A, B) Detection of nucleic acids from various pathogens via ConRCA fluorescence strategy. (C) The ConRCA lateral flow strips readout for the detection of different pathogens. (D) The ConRCA lateral flow strips readout for the band intensity of different pathogens. C: Control line, T: Test line, NC: Negative control.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/fb86520418a8136a0da41e97.png"},{"id":80921752,"identity":"5c471630-5622-40cf-a96f-d7cdde41b933","added_by":"auto","created_at":"2025-04-18 20:56:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9608830,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of various methods for detecting HPV16 in clinical samples. (A) qPCR, ConRCA fluorescence and lateral flow strips strategy for HPV16 clinical samples. (B) Comparison between the ConRCA and the qPCR for clinical samples. (C) ROC curves of the pathogens detected using the ConRCA.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/66f44f67ea97b6659739a504.png"},{"id":83068014,"identity":"bad5e331-855d-463e-8627-c61a629bf714","added_by":"auto","created_at":"2025-05-19 16:09:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":40565770,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/70be4b10-3ac3-4157-9006-69c58e43d91a.pdf"},{"id":80921747,"identity":"b015421d-e389-4259-a85e-6166e441aa3d","added_by":"auto","created_at":"2025-04-18 20:56:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":366735,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/0b2bbb066b959e1ad9791419.docx"},{"id":80921748,"identity":"95084bfc-615f-4cda-af71-2b255d310600","added_by":"auto","created_at":"2025-04-18 20:56:38","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":366735,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedSupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/bb140b631c2f0cf48c4f798b.docx"},{"id":80921447,"identity":"2686ce9c-af51-4530-9cf8-85ea3b93e43a","added_by":"auto","created_at":"2025-04-18 20:48:38","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1538014,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6278232/v1/445c29cae00e5e3c3f80b93f.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Concanavalin A-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay for rapid detection of HPV16","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman papillomavirus (HPV) infection is a global health concern. HPV is an enveloped double-stranded DNA virus that exclusively infects humans, and is responsible for 95% of cervical cancers. It is also the causative agent in approximately 5% of human cancers, including anal, vaginal, vulvar, and penile cancers \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. HPV16 is the most important subtype of the 12 high-risk carcinogenic subtypes, and it is associated with 62.4% of cervical cancers \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Therefore, screening for high-risk subtypes, particularly HPV16, and long-term monitoring are crucial measures for preventing the occurrence of cervical cancer. Currently, polymerase chain reaction (PCR) is a widely used HPV detection technique, known for its high sensitivity and reproducibility \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. However, due to regional conditions, limited technical personnel, and the restriction of detection equipment to specialized laboratories, the coverage and follow-up rates of HPV screening are insufficient \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Therefore, the development and implementation of point-of-care testing technologies with broader applicability, to promote pre-hospital HPV screening, are important measures for improving the quality and coverage of cervical cancer screening.\u003c/p\u003e \u003cp\u003eThe clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system was initially discovered as an adaptive immune mechanism in bacteria. Today, it has demonstrated strong competitiveness in the fields of gene editing and pathogen diagnosis. In particular, some Cas proteins with special functions in this system, such as Cas12 and Cas13, can recognize target sequences and initiate targeted cleavage activity under the guidance of guide RNA (gRNA). These proteins are not only capable of cleaving target DNA or RNA, but also have \u0026lsquo;trans-cleavage\u0026rsquo; abilities, meaning that they can cleave other nontargeted DNA or RNA molecules. By combining reporter nucleic acid molecules labeled with fluorescent groups and utilizing the trans-cleavage properties of Cas proteins, the detection of target genes can be achieved \u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The recombinase\u0026ndash;polymerase amplification (RPA)\u0026ndash;CRISPR/Cas12a method combines the high specificity of CRISPR/Cas12a with the high sensitivity of RPA, allowing the efficient amplification, recognition and cleavage of nucleic acid target sequences in a short time, and significantly enhancing the detection sensitivity. This method has been widely used to analyze the nucleic acids of pathogenic microorganisms, such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e, and SARS-CoV-2 \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, traditional RPA\u0026ndash;CRISPR/Cas12a detection techniques typically require two separate reaction steps, nucleic acid amplification and CRISPR-mediated signal detection. This stepwise operation is relatively cumbersome, increasing the complexity and duration of the experiment. Moreover, the process of transferring amplified products increases the likelihood aerosol cross-contamination and false-positive results. These issues make the technique less than ideal for practical applications, especially in scenarios requiring high-throughput and rapid detection \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. So far, many studies have aimed to integrate the two parts within a single tube using physical separation. This strategy involves adding nucleic acid amplification and CRISPR detection components to the bottom and the wall of the reaction tube, respectively. After the amplification process is completed, mixing is achieved with centrifugation \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Tan et al \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e also reported a one-pot method in which the RPA and CRISPR/Cas12a reactions were separated by paraffin, creating an independent platform upon which the RPA reaction generates sufficient target product before its combination with the CRISPR/Cas12a system. A light-controlled one-pot method for activating Cas12a was also developed to enhance the accuracy and sensitivity of the technique \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. These methods effectively address the issue of aerosol contamination, but they often require additional centrifugation or activation steps, which increase the complexity and cost of the detection process. Overall, achieving the efficient integration of nucleic acid amplification with the CRISPR/Cas system within a single reaction tube still presents technical challenges, limiting its potential application to rapid on-site testing.\u003c/p\u003e \u003cp\u003eSample preprocessing is also a significant factor limiting on-site application. Two commonly used nucleic acid extraction methods, column- and magnetic-bead-based methods, can provide high-purity nucleic acid molecules, but they require repeated pipetting and centrifugation, which are inconvenient operations. Moreover, the need to repeatedly open and close the tubes during nucleic acid extraction can lead to cross-contamination. In recent years, to overcome the drawbacks of traditional nucleic acid extraction methods, various simplified nucleic acid extraction schemes have been proposed. These methods often involve direct lysis techniques that depend on chemical methods, enzymatic methods, or high temperatures. However, for samples with low-concentration targets, these methods can further reduce the probability of isolating the target nucleic acids, thereby reducing the sensitivity of the detection \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. These direct lysis methods also involve a large number of complex components, which may inhibit subsequent amplification and signal readout. Therefore, obtaining target nucleic acids with high purity in a rapid, simple, and efficient manner is a challenge currently faced in sample preprocessing.\u003c/p\u003e \u003cp\u003eLectins are a group of heterogeneous proteins widely found in various plants, invertebrates, and higher animals. They possess specific recognition regions (carbohydrate recognition domains) that can identify and reversibly bind to specific carbohydrates \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Concanavalin A (ConA) is a lectin extracted from leguminous plants, which was among the earliest to be characterized and extensively studied. Research has shown that, in addition to glucose and mannose, which significantly inhibit the hemagglutination activity of ConA, galactose, lactose, trehalose, rhamnose, N-acetyllactosamine, N-acetylglucosamine, sucrose, and maltose also exert inhibitory effects on the hemagglutination activity of ConA \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. This indicates that ConA has a high affinity for these sugar molecules. Therefore, it can be used in the separation and purification of glycosylated substances and viruses, and in the field of biological diagnostics \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Several studies have reported the use of ConA in pathogen detection. Kim \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e developed diagnostic kits for norovirus in which ConA was used to concentrate norovirus. Zhang \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e demonstrated that ConA-modified biofunctional nanoparticles have a minimum limit of detection (LOD) 50 CFU/mL for Gram-positive bacteria. ConA binds to the glycosylation sites of human immunodeficiency virus (HIV) gp120 and overcomes strain limitations \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. This offers a new direction for the development of detection tools for HPV. Another study demonstrated the glycosylation modification of the HPV L1 protein, which ensured the correct folding and structural stability of the protein \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. A recent study reported that after HPV infection, the level of O-GlcNAc glycosylation in cells increases, and that the sugar structure involved in this glycosylation modification is mainly O-linked β-N-acetylglucosamine (O-GlcNAc). This modification involves the attachment of a single N-acetylglucosamine (GlcNAc) residue to a serine (Ser) or threonine (Thr) residue of proteins via a β-glycosidic bond \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. These finds provides inspiration for the detection of HPV based on ConA.\u003c/p\u003e \u003cp\u003eIn this study, we developed a ConA-assisted extraction-free one-pot RPA\u0026ndash;CRISPR/Cas12a assay (ConRCA) for the point-of-care detection of HPV. This method utilizes ConA-coated magnetic beads (ConA MBs) for viral pre-enrichment and combines RPA and the CRISPR/Cas12a recognition amplification system in a single reaction tube. This approach increases the sample concentration and significantly reduces the aerosol contamination caused by repeatedly opening and closing the tube, thus reducing the operational complexity. The results are visualized with fluorescence or lateral flow strips, allowing the visual detection of HPV16 within 25 min. The assay is characterized by its high sensitivity, high specificity, and strong versatility, and provides a reliable and effective technique for the rapid diagnosis and long-term monitoring of HPV patients.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe hiScribe T7 quick high yield RNA synthesis kit, Lba Cas12a, 10\u0026times; NEBuffers 2.1 and RNase inhibitor was obtained from New England Biolabs (Beijing, China). The HPV plasmid, primers, and ssDNA reporters (single-strand DNA reporters, FAM-TTATT-BHQ1 and FAM-TTATTATT-Biotin) were synthesized by Sangon Biotech (Shanghai, China). The DNA isothermal rapid amplification kit was purchased from Amp Future Bio-Tech Co (Changzhou, China). Lateral flow readout strip were purchased from Tolo Biotech. (Shanghai, China). High risk human papillomavirus (HPV) nucleic acid assay kit (qPCR assay) from Liferiver (Shanghai, China). The \u003cem\u003eEasyPure\u003c/em\u003e\u0026reg; RNA kit purchased TransGen Biotech (Beijing, China). ConA MBs were obtained from Biyun Tian Biotechnology Co., Ltd (Shanghai, China). Pathogenic microorganisms DNA/RNA extraction kit was obtained from Beaver (Suzhou, China). Nucleic acid releasing agent was obtained from Amp Future Bio-Tech Co (Changzhou, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of single guide RNA\u003c/h3\u003e\n\u003cp\u003eThe DNA template for in vitro transcription is two reverse complementary paired oligos containing a T7 promoter sequence and spacer sequence. The two DNA templates were annealed at 85\u0026deg;C to 25\u0026deg;C. The annealed product was incubated with T7 RNA polymerase for in vitro transcription at 37\u0026deg;C for 3h. The in vitro transcription reaction was treated with DNase I (Promega) for 10 min at 37\u0026deg;C, and then purified using the The \u003cem\u003eEasyPure\u003c/em\u003e\u0026reg; RNA Kit. The purified sgRNA is stored at -80\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eDesign and screening of RPA primers and sgRNAs\u003c/h3\u003e\n\u003cp\u003eThe six specific RPA amplification primers and eight specific sgRNAs were designed (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) using SnapGene 6.0.2 software. Three upstream primers and two downstream primers in two-by-two combinations totaled six sets. The eight sgRNAs contained four classical protospacer-adjacent motif (PAM) (TTTN) site sgRNAs and four suboptimal PAM (VTTV) site sgRNAs. The RPA products were screened for RPA primers by 1.5% agarose gel electrophoresis, and then the screened RPA primer sets were using CRSIPR/Cas12a fluorescence assay to select sgRNAs.\u003c/p\u003e\n\u003ch3\u003eRPA and qPCR assay\u003c/h3\u003e\n\u003cp\u003eStandard RPA reactions were conducted according to the DNA isothermal rapid amplification kit\u0026rsquo;s protocol. The RPA amplification mix consisted of 29.4 \u0026micro;L of A Buffer, 10 \u0026micro;M each of forward and reverse primers, 2.5 \u0026micro;L of A Buffer, 2 \u0026micro;L of DNA template, and ddH\u003csub\u003e2\u003c/sub\u003eO to obtain a final volume of 50 \u0026micro;L. The reactions were performed at 37\u0026deg;C for 30 min. The HPV qPCR assay were performed in 40 \u0026micro;L reaction volumes containing 36 \u0026micro;L of qPCR mixture and 4 \u0026micro;L lysed sample. The reaction by incubating at 94\u0026deg;C for 2 min, followed by 40 cycles of 93\u0026deg;C for 10 s, 62\u0026deg;C for 31 s.\u003c/p\u003e\n\u003ch3\u003eConRCA systems\u003c/h3\u003e\n\u003cp\u003eOne-pot reactions systems were performed in 40 \u0026micro;L reaction volumes containing 25\u0026thinsp;~\u0026thinsp;100 nM LbCas12a, ssDNA reporter was 400\u0026thinsp;~\u0026thinsp;1000 nM (FAM-TTATT-BHQ1) or 25\u0026thinsp;~\u0026thinsp;50 nM (FAM-TTATTATT-Biotin), 25\u0026thinsp;~\u0026thinsp;200 nM sgRNA, 0.25\u0026times; ~2\u0026times; NEBuffer 2.1, 18 \u0026micro;L RPA mixture. The 2.5 \u0026micro;L B Buffer and 8 \u0026micro;L tested sample were added to each one-pot reaction systems. The readout through SpectraMax i7x or lateral flow strip at 37\u0026deg;C for 15\u0026ndash;60 min.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConA enrichment and HPV DNA extraction\u003c/h2\u003e \u003cp\u003eTo simulate real detection scenarios, a sample pool was created by combining 1 mL each of 10 HPV16-positive samples, generating a 10 mL bulk sample. Enrichment analyses were conducted with 200 \u0026micro;L aliquots, to which another 200 \u0026micro;L of 2\u0026times; binding buffer (40 mM HEPES [pH7.4], 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM MnCl\u003csub\u003e2\u003c/sub\u003e, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e) was added. Each 400 \u0026micro;L sample was spiked with ConA MBs at 0.5, 1, 3, 5, 10, or 15 \u0026micro;L of the total sample volume and incubated at room temperature for 1, 5, 10, 15, 20, 25, or 30 min. The ConA MBs were magnetically pelleted and the supernatant was removed. The ConA MBs were resuspended in 50 \u0026micro;L of nucleic-acid-releasing agent before incubation for 5 min at 100\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe appropriate volumes of these positive pools were then combined with negative clinical samples diluted 1:10 and 1:100 for the recovery experiment. The original samples and diluted samples were each divided into two equal portions. The DNA in one was directly extracted with a commercial nucleic acid extraction kit and the products were quantified with quantitative PCR (qPCR). The other portion was enriched with ConA MBs before all of the mixture was subjected to nucleic acid extraction and the products were quantified with qPCR. Nucleic acid extraction was performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCollection of HPV16 clinical samples\u003c/h3\u003e\n\u003cp\u003eHPV samples were collected in March\u0026ndash;September 2024 at the PLA General Hospital Fourth Medical Center, Beijing, China. The sample swabs were placed in a centrifuge tube containing 1 mL of saline. The processing of clinical samples was performed in accordance with the Materials and Methods section 2.6.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePrinciple of ConRCA\u003c/h2\u003e \u003cp\u003eThe design and reaction mechanism of ConRCA are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This strategy utilizes ConA MBs to rapidly enrich the target and release the nucleic acids within 10 min, avoiding the cumbersome sample pretreatment process required in conventional methods. The release of viral DNA was accomplished with a nucleic-acid-releasing agent. This one-step extraction method achieves effective enrichment of low-concentration targets and the rapid release of nucleic acids, eliminating the need for time- and labor-intensive column purification and bypassing centrifugation. By designing two types of sgRNAs with canonical or suboptimal PAM sites, the trans-cleavage efficiency of Cas12a/sgRNA was verified. When a fluorescence reader or lateral flow strip was used, ConRCA detected as little as 1.2 copies/\u0026micro;L of HPV DNA within 15 min.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeveral reports have indicated that the collateral cleavage activity of Cas is closely related to sgRNAs with different PAM sites. Given that the ConRCA developed in this study necessitates the synchronization of RPA and Cas12a cleavage, the method is contingent on the slower enzymatic kinetics of Cas12a.\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e First, Cas12a is activated using the rapidly accumulating amplicons in the pre-reaction stage. The single-stranded DNA (ssDNA) probe is then sheared by a large number of activated Cas12a. Finally, the rapidly accumulating probe signals are read. Notably, the method does not require a complex way to physically isolate the RPA and CRISPR/Cas reaction systems. The simplicity, speed, and stability of the method makes it suitable for on-site nucleic acid testing, and obviates the need for specialized laboratories and healthcare professionals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of ConA-assisted enrichment conditions\u003c/h2\u003e \u003cp\u003eThe parameters for target enrichment, including the input volume of ConA MBs (10 mg/mL) and the incubation time, were optimized to minimize the resultant cycle threshold (Ct) values. First, parallel aliquots (200 \u0026micro;L) of 10 positive clinical samples mixing were analyzed with volumes of ConA MB of 0.5\u0026ndash;15 \u0026micro;L. The optimal ConA MB input volume was determined to be 10 \u0026micro;L, because 0.5\u0026ndash;5 \u0026micro;L and 15 \u0026micro;L produced higher relative Ct values, indicating lower DNA concentrations after extraction, whereas an input volume of 10 \u0026micro;L produced the lowest Ct value (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This ConA MB input volume was used to determine the optimal incubation time in the range of 1\u0026ndash;30 min. The ideal incubation time was determined to be 5 min. The highest Ct value was observed at 1 min, and the values tended to converge after 5 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the efficacy of ConA-mediated target enrichment, we conducted tests on two 200 \u0026micro;L samples with identical dilution factors, as detailed in the Materials and methods section 2.6. Specifically, one sample was subjected to commercial nucleic acid extraction followed by qPCR to determine the Ct value. The other sample was enriched with ConA, and the supernatant was discarded after magnetic selection. The magnetic beads were then resuspended in 200 \u0026micro;L of nuclease-free water. This resuspended mixture was used as the new sample for commercial nucleic acid extraction and subsequent qPCR analysis. This procedure was designed to negate any variability introduced by the nucleic acid extraction process, thus providing a more-precise evaluation of the enrichment efficiency. Two-way ANOVA with replication revealed no significant difference between the means (α\u0026thinsp;=\u0026thinsp;0.05, P\u0026thinsp;=\u0026thinsp;0.2238), indicating that the Ct values obtained during DNA amplification with both methods were statistically indistinguishable across all dilutions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). This finding suggests that the ConA-assisted assay gives essentially the same results as the conventional nucleic acid extraction method at the concentrations tested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePrimer and sgRNA screening\u003c/h2\u003e \u003cp\u003eSix pairs of specific RPA primers were designed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Supplementary Table\u0026nbsp;1). Including three forward primers and two reverse primers, which were paired in all possible combinations, resulting in a total of six different primer pairs. Agarose gel electrophoresis of the RPA products showed that when the concentration of the amplification template was 1.2 \u0026times; 10\u0026sup3; copies/\u0026micro;L, the primer combinations F1R1, F2R1, F3R1, and F3R2 all successfully amplified the target fragment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The target band (230 bp) amplified by the primer combination F3R1 was brightest. Therefore, the optimal primer combination F3R1 was selected for the subsequent one-pot reaction systems examined in this study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the conserved sequences in the F3R1 primers, eight sgRNAs with different PAM sites (including the classic TTTN and suboptimal VTTV motifs) were designed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In the one-pot reaction system, sgRNA and Cas12a acted as the guide RNA and nuclease, respectively, to form a ribonucleoprotein complex (RNP). We then investigated the cleavage effects of the RNPs formed by two sgRNAs with different PAM sites and Cas12a. In this study, two-step and one-pot assays for fluorescence detection were used to screen the sgRNAs. The result of the two-step assay showed that canonical sgRNA1 and sgRNA4 both produced fluorescent signals, and that canonical sgRNA1 had the highest relative fluorescence value (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The results of the one-pot assay showed that all four suboptimal sgRNAs produced fluorescent signals, and that suboptimal sgRNA1 had the highest relative fluorescence value (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo compare the effects of the optimal canonical sgRNA and suboptimal sgRNA on the cleavage efficiency of Cas12a, we used both two-step and one-pot assays with the selected canonical sgRNA1 and suboptimal sgRNA1. The results indicated that in the two-step assay, canonical sgRNA1 yielded a higher fluorescent detection value than suboptimal sgRNA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In contrast, in the one-pot assay, suboptimal sgRNA1 demonstrated an earlier onset of fluorescence and a higher fluorescence value than canonical sgRNA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These findings are consistent with previous studies that have explored the influence of different PAM sites on Cas12a activity.\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e It has been demonstrated in previous studies that the design of sgRNA based on different PAM sites directly affects the cleavage efficiency of Cas12a. It is noteworthy that within one-pot reaction systems, Cas12a guided by a suboptimal sgRNA shows better cleavage efficiency than Cas12a guided by canonical sgRNA \u003csup\u003e[\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. These findings suggest that the use of suboptimal sgRNA in one-pot reaction systems allows the acquisition of results in as little as 15 min, without compromising the detection accuracy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). This approach has been shown to significantly reduce the detection time and simplify the procedure, thereby minimizing the risk of aerosol nucleic acid contamination. Consequently, suboptimal sgRNA1 was selected for subsequent experiments in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of CRISPR reaction parameters\u003c/h2\u003e \u003cp\u003eTo enhance the cost-effectiveness and performance of the one-pot reaction system, we optimized the working concentrations of critical components within the reaction system. These components included Cas12a, the ssDNA probe, NEBuffer 2.1, and sgRNA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the detected fluorescence values were significantly higher when Cas12a was at a concentration of 50 nM than at 25 nM or 100 nM. The lower fluorescence value at 100 nM may be attributable to the glycerol concentration in the protein preservation solution. There are many free molecules in one-pot reaction systems, and higher concentrations of glycerol will affect the free state of each molecule, which in turn will affect the reaction efficiency. Therefore, the optimal working concentration of Cas12a was selected as 50 nM in this study. We next tested the effect of different concentrations of the ssDNA fluorescent probe. Previous studies have reported Cas12a to ssDNA ratios of 1:7 or 1:10 \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the detected fluorescence values gradually increased as the working concentration of the ssDNA fluorescent probe increased. When the concentration of the ssDNA probe increased from 800 nM to 1 \u0026micro;M, there was a noticeable acceleration in the rate of fluorescence increase. Consequently, 1 \u0026micro;M was selected as the optimal working concentration of the ssDNA probe. The ratio of Cas12a to ssDNA was 1:4. We next optimized the amount of NEBuffer 2.1 added to the reaction system. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026times; NEBuffer 2.1 produced the lowest detected fluorescence value, and 0.25 \u0026times; NEBuffer 2.1 produced the highest detected fluorescence value. The reason may be that the high concentration of salt ions had some inhibitory effect on RPA. Therefore, we selected the optimal working concentration as 0.25 \u0026times; NEBuffer 2.1. Finally, we optimized the sgRNA concentration, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD. The highest fluorescence value was detected when the working concentration of sgRNA was 100 nM, which selected as the optimal working concentration. In summary, the optimized working concentration ratio was Cas12a: sgRNA: ssDNA\u0026thinsp;=\u0026thinsp;1:2:4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSensitivity and specificity of ConRCA\u003c/h2\u003e \u003cp\u003eA gradient dilution of the HPV16 DNA plasmid (1.2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, 1.2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, 1.2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e, 1.2 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e, 1.2 \u0026times; 10\u003csup\u003e1\u003c/sup\u003e, 1.2 \u0026times; 10\u003csup\u003e0\u003c/sup\u003e, 1.2 \u0026times; 10\u003csup\u003e\u0026minus;1\u003c/sup\u003e copies/\u0026micro;L) was used for the qPCR assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the LOD of the qPCR was 1.2 \u0026times; 10\u003csup\u003e0\u003c/sup\u003e copies/\u0026micro;L, whereas 1.2 \u0026times; 10\u003csup\u003e\u0026minus;1\u003c/sup\u003e copies/\u0026micro;L was not detected. The sensitivity of the fluorescence method and lateral flow strip was evaluated with ConRCA. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;F, the sensitivity of the fluorescence and lateral flow strip methods was 1.2 \u0026times; 10\u003csup\u003e0\u003c/sup\u003e copies/\u0026micro;L, consistent with the qPCR results. Notably, the ConRCA reaction took only 15 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Compared with the detection times documented in previous studies (85 min, 60 min, and 45 min), the operation was more expeditious and efficient in this study. This demonstrates the potential for the development of on-site detection or at-home self-testing technologies \u003csup\u003e[\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the specificity of ConRCA, we tested six nucleic acid templates: HPV16, human immunodeficiency virus (HIV), hepatitis B virus (HBV), HPV18, HPV6, and HPV11. The test was performed with the ConRCA fluorescence readout, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B. This method specifically detected HPV16, with no obvious cross-reaction with the other pathogens tested. The test was then performed with the ConRCA lateral flow strips readout, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D. The test strips specifically showed HPV16-positive bands after a 15 min reaction time, with no obvious cross-reactivity with the other pathogens tested, and the results were consistent with those of the fluorescence method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eExamination of the clinical samples\u003c/h2\u003e \u003cp\u003eThe vaginal secretions from HPV-infected patients are characterized by their diversity and complexity, posing a challenge to existing methods of rapid HPV nucleic acid testing \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. In this study, we developed a simple sample pretreatment technique using ConA-MBs, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, which achieved efficient nucleic acid release within 10 min.\u003c/p\u003e \u003cp\u003eTo evaluate the performance of ConRCA in detecting HPV16 in clinical samples, 31 clinical samples were collected, including 21 HPV16-positive samples with different viral loads (sample 1\u0026ndash;21) and 10 HPV-negative samples (sample 22\u0026ndash;31). The qPCR method was first used to analyze these samples. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, the positive detection rate for the qPCR method was 95% (20/21). The result for sample 6 was negative, which could be attributed to the degradation of the nucleic acids during storage and transportation. ConRCA was then used to evaluate the same samples with a fluorescence reader or lateral flow strip. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, sample 6 was also negative with both the fluorescence and the strip test, consistent with the results of qPCR. Notably, the result for sample 15 using the strip test was negative (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The Ct value of 38.12 for sample 15 and the low relative fluorescence value detected with the ConRCA fluorescence strategy suggest that the sensitivity of the test strip may be slightly lower than that of the ConRCA fluorescence strategy and qPCR in clinical samples, which is in general agreement with the results of a previous study \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the results of the three methods described above, the nucleic acids in sample 6 had been degraded, leading to a negative result. Using the qPCR results as the gold standard, the sensitivity of the ConRCA fluorescence strategy was calculated to be 100% (20/20) and the specificity was also 100% (11/11) when ConRCA was used to test clinical samples. The sensitivity of the lateral flow strip method was 95% (19/20) and the specificity was 100% (11/11). The overall sensitivity was 95% (19/20) and the specificity was 100% (11/11) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). From the receiver operating characteristic (ROC) curve obtained from the statistical analysis shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, the area under the curve (AUC) for ConRCA was 0.9722 (95% confidence interval: 0.9243\u0026ndash;1.0000). These findings demonstrate the efficacy of the established method in accurately and reliably detecting HPV16 in actual samples.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we have developed a ConA-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay, designated ConRCA, for the rapid on-site detection of HPV16. The results can be evaluated with a fluorescence reader or lateral flow strip within 25 min. The LOD for HPV16 DNA is 1.2 copies/\u0026micro;L. The results for 31 clinical samples demonstrated that ConRCA has a sensitivity of 95.00% and a specificity of 100.00%, which is consistent with the qPCR technology. ConRCA allows rapid and sensitive testing with simple operation, offering significant advantages for on-site diagnosis. Furthermore, the development of multichannel microfluidic chips that integrate primers and sgRNAs specific to various HPV types has potential utility in high-throughput HPV screening and at-home self-examination.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYou Nie:\u003c/strong\u003e Writing – original draft, Visualization, Validation, Methodology, Investigation, Conceptualization. \u003cstrong\u003eXiaohui Li:\u003c/strong\u003e Writing – original draft, Validation, Methodology, Investigation, Conceptualization. \u003cstrong\u003eWen Yang:\u003c/strong\u003e Writing – original draft, Visualization, Validation, Methodology, Investigation. \u003cstrong\u003eSihan Fei:\u003c/strong\u003e Collection of clinical samples. \u003cstrong\u003eYingfan Wang:\u003c/strong\u003e Resources, Data curation. \u003cstrong\u003eYazhuo Li:\u003c/strong\u003e Software, Data curation. \u003cstrong\u003eKe Zhang:\u003c/strong\u003e Software, Data curation. \u003cstrong\u003eJiarui Kang:\u003c/strong\u003e Software, Data curation. \u003cstrong\u003eYang Cheng:\u003c/strong\u003e Formal analysis, Data curation. \u003cstrong\u003eHongwei Wang:\u003c/strong\u003e Writing – review \u0026amp; editing, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. \u003cstrong\u003eDandan Liu:\u003c/strong\u003e Writing – review \u0026amp; editing, Validation, Supervision, Methodology, Investigation, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Youth Independent Innovation Science Foundation of the General Hospital of the Chinese People's Liberation Army (grant number 22QNFC098) and the Military Family Planning Program of China (grant number 24JSZ12).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edeclaration\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the ethics committee of the PLA General Hospital Fourth Medical Center in accordance to the Helsinki Declaration (S2021-564-01).\u0026nbsp;All the procedures were performed in accordance with the Declaration of Helsinki, and the patient’s confidentiality was maintained throughout the investigation. All individuals provided their written informed consent to participate in this study. All methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\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"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWilliamson AL (2023) Recent Developments in Human Papillomavirus (HPV) Vaccinology. 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Talanta 278:126449\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Concanavalin A, Extraction-free assay, CRISPR/Cas12a, One-pot detection, HPV","lastPublishedDoi":"10.21203/rs.3.rs-6278232/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6278232/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuman papillomavirus (HPV) infection is a major threat to women\u0026rsquo;s health worldwide. High-risk subtypes, particularly HPV16, require rigorous screening and long-term surveillance to control cervical cancer. However, traditional HPV testing is hampered by the need for nucleic acid extraction, reliance on specialized technicians, and fluorescence detection equipment, limiting its suitability for rapid on-site testing. In this study, we developed a Concanavalin A-assisted extraction-free one-pot recombinase polymerase amplification (RPA) CRISPR/Cas12a assay (ConRCA) for HPV16. Concanavalin A-coated magnetic beads were used for target enrichment and nucleic-acid-extraction-free processing. Suboptimal protospacer-adjacent motifs were used to achieve a one-pot RPA\u0026ndash;CRISPR/Cas12a assay. The ConRCA assay can be completed in approximately 25 min under isothermal conditions and can detect at least 1.2 copies/\u0026micro;L of HPV16 genomic DNA using a fluorescence reader or test strip. The feasibility of this detection method was evaluated with 31 unextracted clinical samples. Compared with qPCR, the overall sensitivity was 95% (19/20), and the specificity was 100% (11/11). Our results indicate that the ConRCA assay has great potential utility as a point-of-care testing for the rapid identification of HPV.\u003c/p\u003e","manuscriptTitle":"Concanavalin A-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay for rapid detection of HPV16","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-18 20:48:33","doi":"10.21203/rs.3.rs-6278232/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-08T15:18:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-07T19:15:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-07T03:43:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-04T12:53:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290084542899834134949472580976308837481","date":"2025-03-31T08:03:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211838150468858837685851387036077190949","date":"2025-03-31T00:57:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66249138414599705248949230933833785402","date":"2025-03-30T20:34:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-30T17:53:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-26T17:15:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-26T01:33:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-03-21T13:51:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"60ed3a2e-3335-4405-903f-488229062ed2","owner":[],"postedDate":"April 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:04:19+00:00","versionOfRecord":{"articleIdentity":"rs-6278232","link":"https://doi.org/10.1007/s00604-025-07198-7","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2025-05-14 15:57:43","publishedOnDateReadable":"May 14th, 2025"},"versionCreatedAt":"2025-04-18 20:48:33","video":"","vorDoi":"10.1007/s00604-025-07198-7","vorDoiUrl":"https://doi.org/10.1007/s00604-025-07198-7","workflowStages":[]},"version":"v1","identity":"rs-6278232","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6278232","identity":"rs-6278232","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}