{"paper_id":"1d64f604-c9f5-4579-836a-e39fc70cf931","body_text":"Proximity Ligation Mediated Signal Recycling with Primer Exchange Reaction for Sensitive and Accurate Methicillin-Resistant Staphylococcus Aureus (MRSA) Detection | 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 Proximity Ligation Mediated Signal Recycling with Primer Exchange Reaction for Sensitive and Accurate Methicillin-Resistant Staphylococcus Aureus (MRSA) Detection Huali Xu, Xiangke Yang, Wen Wang, Xiaomin Yuan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4505973/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Infectious diseases have emerged as a significant global concern, posing a substantial burden in terms of the high morbidity and mortality, and presenting considerable challenges in clinical diagnosis and treatment. Therefore, it is highly-desired to develop new strategies for sensitive and accurate bacteria detection to address the global epidemic of antibiotic resistance. Results In this study, a new technique combining proximity ligation and primer exchange reaction (PER) was developed for precise identification and highly sensitive detection of Methicillin-Resistant Staphylococcus Aureus (MRSA). The antibodies recognize both protein A and PBP2a on the surface of MRSA, leading to the initiation of proximity ligation and PER process. The PER procedure generated a substantial number of G-quadruplex sequences, which subsequently bind with thioflavin T (ThT) to significantly amplify its fluorescence, enabling the detection of MRSA with a low detection limit of 3.5 cfu/mL. Conclusion Due to its non-label format, high selectivity, and sensitivity, this method can serve as a practical and versatile approach for detecting different bacteria in the early stages of infectious diseases. Methicillin-Resistant Staphylococcus Aureus (MRSA) G-quadruplex primer exchange reaction thioflavin T proximity ligation assay Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The prevailing idea that the majority of surgical site infections (SSIs) that arise after elective surgery for liver cancer, following normal methods of antisepsis, are caused by intraoperative contamination, has not been substantiated [ 1 , 2 ]. Bacterial infectious diseases have emerged as a significant worldwide problem, posing a serious threat to human health [ 3 ]. Moreover, increasing evidence suggests that bacteria can also indirectly contribute to the development and advancement of other diseases, including cancer. There has been a rise in drug-resistant bacteria in recent years, resulting in a significant increase in the challenge and expense of developing new effective antibacterial therapeutic strategies. Methicillin-Resistant Staphylococcus Aureus (MRSA) is a prominent contributor to intractable bacterial infections [ 4 – 6 ]. Hence, the accurate and highly sensitive identification of MRSA from clinical samples is crucial for the early detection and treatment of MRSA related infection. The mecA gene mediates methicillin resistance by expressing penicillin binding proteins (PBP) and PBP2a [ 7 , 8 ]. Therefore, the detection of the mecA gene or its product PBP2a is frequently used as an additional biomarker to quickly confirm methicillin resistance. Several MRSA detection approaches have been developed in recent years, primarily categorized into mecA gene sequencing [ 9 ] and membrane protein analysis methods [ 10 – 13 ]. Despite the reliability and accuracy of the procedure, there are still significant deficiencies in these methods. Conventional colony counting techniques, which are commonly employed in clinical laboratories, are criticized by complicated procedures and inability to precisely identify colonies during the first phase of disease and assess treatment resistance [ 14 ]. DNA sensors are highly regarded for the exceptional detection sensitivity; however, they often face criticism due to the possibility of false positive results caused by interfering components present in clinical samples [ 15 – 17 ]. Hence, there is a significant promise in promoting a precise and highly sensitive technique for detecting MRSA. Given that MRSA has the ability to express several types of proteins such as PBP2a and protein A, multiple analytical techniques have been published to measure MRSA levels by identifying a MRSA specific protein. However, proteins released by the cell lysis and MRSA may generate undesired inaccurate signals. To reduce interference, two or more recognition sites are needed simultaneously. The recently developed dual-recognition [ 12 ] mediated MRSA identification strategy has demonstrated promising advancements in detection accuracy, reliability, and low background noise. This strategy involves the simultaneous targeting of two membrane proteins using dual aptamers [ 18 ], dual antibodies [ 19 ], or a combination of aptamer and antibody [ 20 ], as well as aptamer and cholesterol [ 21 ]. Although significant advancements have been made, these techniques may still encounter challenges, such as multiple separation and limited signal amplification efficiency. Therefore, there is still a significant need for MRSA detection methods that are simple, sensitive, and easy-to-operate. The proximity hybridization approach, reliant on simultaneous recognition of the target molecules by both DNA probes, has demonstrated exceptional selectivity. Furthermore, the proximity hybridization product can be additionally integrated with a conventional nucleic acid amplification technique to enhance sensitivity. We assume that the issues may be resolved by combining dual antibody-based target recognition with proximity ligation-mediated signal recycling (Fig. 1 ). This approach specifically targets protein A, a membrane protein that is present in both MRSA, and PBP2a. The PBP2a-specific antibody and protein A-specific antibody are each bound to two distinct nucleic acid sequences to create the probes for proximity ligation (P1 and P2). The P1 and P2 probes consist of four functional sections: “Antibody” for target recognition, “Spacer”, “Complementary”, and “Trigger”. In the presence of MRSA, the P1 and P2 probes specifically attach to the protein A and PBP2a on the surface of MRSA, respectively. This enables the close proximity of the “C1” and “C2” sections, allowing for proximity hybridization. The close connection between the P1 and P2 probes results in the formation of an intact “Trigger sequence”, which can be used to enhance the following signal. Specifically, the “Trigger sequence” unfolds the hairpin probe (HP) to release the toehold portion. The toehold region forms a binding interaction with the “2” region of the primer exchange reaction probe (PER probe). With the aid of DNA polymerase, the chains are elongated using the “toehold” part and “2” section as primers. Consequently, the “Trigger sequence” is released, leading to the formation of a signal cycle. Additionally, a single-stranded DNA (ssDNA) chain containing the transcribed “3*” section is appended to the end of the “toehold” region of HP. The nicking site enzyme recognizes the specific sequence “3*” and creates a nicking site. The elongation of the ssDNA chain is halted by the “Stop site” in the PER probe and competed by the “1” segment. Consequently, a substantial quantity of “4*” sequences are generated, specifically G-quadruplexes. A multitude of G-quadruplex sequences interact with thioflavin T (ThT) to generate substantial fluorescence, enabling the detection of MRSA with excellent sensitivity and precision. 2. Materials and methods 2.1 Chemicals and materials MRSA strain (ATCC 17802), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 19115), and Streptococcus pneumonia (ATCC 50761) were provided by the American Type Culture Collection (ATCC). The design of all oligonucleotides was based on previous studies ( Table S1 ) and they were produced by Shanghai Sangon (Shanghai, China). All of the oligonucleotides were utilized without additional purification. Nb.BbvCI, Bst polymerase, and deoxy-ribonucleoside triphosphate (dNTPs) were obtained from New England Biolabs, Inc. (Beijing, China). Thioflavin T (3,6-dimethyl-2-(4-dimethylaminophenyl)benzo-thiazolium cation, ThT) were obtained from Sigma-Aldrich (Shanghai, China). Diethypyrocarbonate (DEPC)-treated water (DNase, RNase free) obtained from Beyotime Institute of Biotechnology (Shanghai, China) was used in all experiments. More experimental details were shown in electronic supporting information (ESI). 2.2. Bacteria culture The MRSA bacteria were cultured at a temperature of 37°C for a period of 18 hours using a solution containing 3% NaCl. E. Coli and P. aeruginosa were cultivated in Lysogeny Broth (LB) medium for 12 hours at a temperature of 37°C with a rotation speed of 200 rpm. The MRSA bacteria were grown in a 3% NaCl alkaline protein aqueous medium for 18 hours for the CFU test. The cell suspension was serially diluted, followed by 50 µL of each gradient plated in triplicate on a TCBS dish and incubated at 37°C for 24 h. Colonies were then counted and the average of three parallel dishes was recorded. 2.3 Analytical procedure The bacteria, at different concentrations, were incubated for 40 min in 200 µL of binding buffer containing 0.5 µM P2 probe and 1 µM P1 probe, respectively. Subsequently, a hairpin molecule with a concentration of 150 nM and a PER probe with a concentration of 200 nM were introduced into the solution. Subsequently, the resultant combination was held at a temperature of 80°C for a duration of 20 min and then cooled down to 25°C. Subsequently, the combination was incubated with the ThT dye (10 mM) for an additional 30 min and then diluted in Tris-HCl solution to a volume of 200 µL in order to proceed with fluorescence recording. The RF-6000 Shimadzu fluorescence spectrophotometer (Tokyo, Japan) was used to record the fluorescence spectra. 2.4 Statistical data analysis Each test was repeated at least three independent replicates, which were displayed as the mean ± standard deviation (SD). Data were visualized using software GraphPad Prism 8.0 (CA, USA). The Student's t-test was used to analyze comparisons between two groups. Differences were considered significant at values of P < 0.05. 3. Results and discussion 3.1 Feasibility characterization of the method To validate the detection of MRSA using P1 and P2 probes, we labeled the ends of P1 and P2 probes with fluorescent dyes FAM and Cy3, respectively. The P1 and P2 probes were individually exposed to MRSA for a period of 30 min. Subsequently, any probes that did not bind to the MRSA were removed using centrifugation. The fluorescence signal of the sediment is detected and then compared. Figure 2 a exhibits a significant augmentation in the fluorescence signals of FAM and Cy3 when MRSA is present, demonstrating the successful attachment of P1 and P2 probes to MRSA. In order to verify the disassociation of HP using proximity hybridization of P1 and P2 probes, FAM and BHQ are attached to both ends of HP. Figure 2 b shows that the FAM fluorescence signal was significantly reduced when linear HP was folded into a hairpin structure, as a result of the quenching action of BHQ on the FAM signal. When P1 and P2 are present, the fluorescence intensity of HP remains low, indicating that HP is not activated in these circumstances. The fluorescence signal of FAM was significantly enhanced only when MRSA, P1, and P2 were all present simultaneously, indicating the unfolding of the HP hairpin structure. When both MRSA and P1 or MRSA and P2 were simultaneously present, the FAM signals consistently remained at a low level, indicating that the activation of HP need close proximity hybridization between P1 and P2 probe. Multiple samples were prepared using different parameters to evaluate the effectiveness of the proximity hybridization-dependent approach for accurately detecting MRSA. Figure 2 c illustrates that the blank sample shows a diminished level of fluorescence intensity due to the absence of G-quadruplex formation (column 1). Sample 2, which does not include the P1 probe (column 2) and P2 probe (column 3), has a minimal degree of fluorescence, indicating the absence of proximity hybridization in the absence of the probes. Sample 3 (column 4) shows a diminished fluorescence intensity because MRSA is not present, which prevents the occurrence of proximity hybridization-dependent PER response. The fluorescence signal of the sample was much increased when all essential components were included in the sensor system (column 5). 3.2 The optimization of experimental conditions Once the potential of our signal amplification bio-sensing method for detecting MRSA was confirmed, we proceeded to optimize the experimental parameters that influenced the performance of this MRSA detection method. These parameters included the concentration of the HP, the duration of the enzyme reaction, the amount of Bst polymerase, and the concentration of ThT. The optimization findings are displayed in Fig. 3 , where F and F 0 represent the fluorescence intensity of the reaction mixture at 490 nm with and without the presence of 5 × 10 6 cfu/mL of MRSA, respectively. Initially, the HP concentration was examined. As shown in Fig. 3 a, the fluorescence response steadily increases and reaches a plateau at a concentration of 100 nM of HP. Consequently, it was discovered that a concentration of 100 nM of HP is ideal for MRSA detection. Following the optimization of HP concentration, the dosage of Bst polymerase was measured ranging from 3 U/µL to 18 U/µL, with an increase of 3 U/µL. The findings can be seen in Fig. 3 b. The fluorescence intensity rises as the amount of Bst polymerase grows and reaches its highest value at 15 U/µL. Consequently, the concentration of Bst polymerase in following tests was 15 U/µL. Figure 3 c illustrates the correlation between the reaction time of the enzyme and the fluorescence response. The fluorescence response increases significantly within the time frame of 30 min to 120 min, however there is no noticeable change after 120 min. Furthermore, the impact of ThT at various doses on the fluorescence emission intensity of the reaction solution was investigated (Fig. 3 d). Within the range of concentrations examined, the fluorescence intensity increases until it reaches its highest value at 8 mM when the ThT concentration is raised. This indicates that the ideal concentration of ThT is 8 mM. 3.3 Detection of MRSA using the established strategy The proposed technique was utilized for MRSA detection under optimum conditions, taking advantage of its strong potential to produce amplified signal outputs. Initially, we examined its viability. Figure 4 a demonstrates that the fluorescence of the approach was insignificant when MRSA was not present. When MRSA was present at diverse concentrations (ranging from 10 to 10 6 cfu/mL), the fluorescence exhibited a significant increase as the MRSA concentration increased. Furthermore, we observed a direct relationship between the measured fluorescence intensity and the logarithm of MRSA concentration (C) within the range of 10 to 10 6 cfu/mL (Fig. 4 b), which covers a span of six orders of magnitude. The calibration equation can be expressed as: F = 93.64*lgC + 2.940. The correlation coefficient (R 2 ) for this equation is 0.9869, as shown in Fig. 4 c. The exceptional sensitivity is mostly attributed to the simultaneous amplification of signals through both target recycling and PER process. An important obstacle in MRSA analysis is the capacity to precisely distinguish MRSA from a wide range of microorganisms. In this study, the specificity was evaluated using the detection of E. Coli , P. aeruginosa , and S. pneumoniae . The proposed method allows for the detection of these interfering bacteria and their combination. Figure 4 d demonstrates the contrast in fluorescence signals’ reaction to various bacteria. The signal was feeble in the presence of E. Coli , P. aeruginosa , and S. pneumoniae , as opposed to MRSA. In addition, we also utilized the technique to identify a combination of bacteria ( E. coli , P. aeruginosa , S. pneumoniae , and MRSA), resulting in a substantial increase in fluorescence compared to the control group. Presently, point-of-care (POCT) is garnering growing interest in the detection of disease-related targets due to the simple detection procedure and devices. In this process, we combine all the elements of the approach and place them in a microwell plate to create a portable device for detecting MRSA. Subsequently, the microwell plate was employed to identify E. coli , P. aeruginosa , and S. pneumoniae . The fluorescence intensity of these interfering bacteria, MRSA, and their combination was measured using a microplate reader. Figure 4 e demonstrates a comparable fluorescence intensity trend, suggesting that the approach has significant promise for point-of-care testing (POCT) applications. 3.4 Analysis of MRSA from clinical samples Interference in the clinical samples can impede the binding of antibodies to the target protein and disrupt the proximity ligation process. Hence, it was imperative to evaluate the suitability of this approach for clinical sample detection. To replicate the clinical condition, MRSA with varying concentration gradients were introduced into serum. Serum samples were acquired by centrifuging a clinically obtained normal human blood sample. Initially, the amount of MRSA in clinical samples was determined using the conventional colony counting method and the suggested technique (Fig. 5 a). The results obtained from the four clinical samples using the method was in high agreement with those obtained using the traditional colony counting method, demonstrating the reliability of our suggested method. In order to showcase the possible POCT application of the proposed approach, we conducted a test on four clinical samples using a microplate under the optimized experimental conditions. The outcome, as depicted in Fig. 5 b, can specifically distinguish the interfering bacteria from samples, indicating the POCT application potential of the method. 4. Conclusion Efficiently and precisely identifying disease-causing bacteria in clinical samples is a global obstacle. In addition to the conventional colony count approach, two primary methods have been documented for the detection of bacteria: aptamer-based detection method and protein detection method. Generally, aptamer-based detection methods have been found to be more sensitive because aptamers may convert the levels of membrane protein into nucleic acid detection. On the other hand, protein detection methods assure high specificity by relying on the interaction between antigen and antibody. Nevertheless, the current methods for detecting MRSA still encounter the following issues: i) The existing MRSA detection methods lack enough sensitivity for trace amount MRSA detection; ii) The current MRSA detection technologies is not suitable for POCT applications; iii) The detection outcome is highly susceptible to interference from a single protein on the surface of MRSA. In this study, we present a novel method for detecting MRSA by combining the benefits of proximity ligation and PER procedure. We devised a sophisticated approach to concurrently identify the presence of protein A and PBP2a. When combined with a dual signal amplification, our findings demonstrate that MRSA can be accurately and directly detected with a high level of sensitivity. By implementing the proposed method in POCT, we anticipate its versatility in detecting various bacteria, with exceptional specificity and quick amplification. Declarations Supplementary Information The online version contains supplementary material available at: xxx-xxx-xx. Acknowledgements We appreciate the financial support from the Jiaozuo People’s Hospital. We also thank the director of central laboratory for providing essential equipment. Author contributions The study was conceived by Huali Xu. Huali Xu conducted the lab work and wrote the manuscript. Xiangke Yang, Wen Wang, and Xiaomin Yuan assisted data analysis. All authors discussed and aided in interpreting the results. Data Availability All data generated and analyzed during this study are included in this article. Ethical Approval This article does not contain any studies with human participants or animals performed by any of the authors. Consent to Participate Not applicable. Consent for Publication Not applicable. Conflict of Interest The authors declare no competing interests. Fund: Not available References Seidelman J, Anderson DJ: Surgical Site Infections. Infect Dis Clin North Am 2021, 35(4):901-929. Seidelman JL, Mantyh CR, Anderson DJ: Surgical Site Infection Prevention: A Review. JAMA 2023, 329(3):244-252. Liu C, Bayer A, Cosgrove SE, Daum RS, Fridkin SK, Gorwitz RJ, Kaplan SL, Karchmer AW, Levine DP, Murray BE et al : Clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children. Clin Infect Dis 2011, 52(3):e18-55. Burnham CD, Leeds J, Nordmann P, O'Grady J, Patel J: Diagnosing antimicrobial resistance. Nat Rev Microbiol 2017, 15(11):697-703. 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Su J, Zheng W, Pan Y: Proximity ligation initiated DNAzyme-powered catalytic hairpin assembly for sensitive and accurate microRNA analysis. Anal Biochem 2023, 680:115299. Wang P, Yang Y, Hong T, Zhu G: Proximity ligation assay: an ultrasensitive method for protein quantification and its applications in pathogen detection. Appl Microbiol Biotechnol 2021, 105(3):923-935. Greenwood C, Johnson G, Dhillon HS, Bustin S: Recent progress in developing proximity ligation assays for pathogen detection. Expert Rev Mol Diagn 2015, 15(7):861-867. Zhao X, Luo C, Mei Q, Zhang H, Zhang W, Su D, Fu W, Luo Y: Aptamer-Cholesterol-Mediated Proximity Ligation Assay for Accurate Identification of Exosomes. Anal Chem 2020, 92(7):5411-5418. Additional Declarations No competing interests reported. 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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-4505973\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":314038304,\"identity\":\"8fbac9d5-8126-4304-a106-ec9fdc32392a\",\"order_by\":0,\"name\":\"Huali Xu\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3PsWrDMBCA4TMCTWq1niEkr3Al4CkPc8bgyYGMHgI1pCRDSLymb5Gxo4JBWeTdY/wEbbZ2KX2AlsjdMuib7+fuAILgDkld9/1Hic/15L2/cLn0J4/oxBTdLHqtCkEXZ/3JGAqJD+s8OppCxv2LGHAYtAZRNiKuWlumlQS92fLtROyZFqqROtrnXfo2AnTt0bPFECM2Kl5B0qVOAuHclzAZRQ2ShWSRrsWQpHiqFOdETiUwLEGbCTQzjg8yQ3ZWeX+Z1Kvz1/UbWaM4XT/L5VhvdreTX9T/xoMgCII//QAnVEoWxd4FGAAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Jiaozuo People’s Hospital\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Huali\",\"middleName\":\"\",\"lastName\":\"Xu\",\"suffix\":\"\"},{\"id\":314038306,\"identity\":\"f6cb9eb2-e4f6-4f36-907e-0baf9412b91b\",\"order_by\":1,\"name\":\"Xiangke Yang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"The Third People’s Hospital of Jiaozuo\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiangke\",\"middleName\":\"\",\"lastName\":\"Yang\",\"suffix\":\"\"},{\"id\":314038308,\"identity\":\"ccc4887b-a12f-4344-ac47-8a59f9694ba2\",\"order_by\":2,\"name\":\"Wen Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiaozuo People’s Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Wen\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":314038314,\"identity\":\"fa1ed024-639f-413f-9e4e-a98bbc8c7868\",\"order_by\":3,\"name\":\"Xiaomin Yuan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jiaozuo People’s Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiaomin\",\"middleName\":\"\",\"lastName\":\"Yuan\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-05-31 03:38:32\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4505973/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4505973/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":58335445,\"identity\":\"553daec4-f66a-4e27-9815-043df4f3eaa7\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 05:20:15\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":126388,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMechanism description of the proposed sensitive and label-free amplification method for the detection of MRSA via target recycling and PER amplifications\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4505973/v1/88d88bb5bc9db72d30e0a447.png\"},{\"id\":58336266,\"identity\":\"becd822b-1a05-4510-8a39-19a4eabe68a3\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 05:28:15\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":163847,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFeasibility analysis of the method for MRSA detection. \\u003c/strong\\u003e(a) Fluorescence intensity of the FAM and Cy3 labeled P1 and P2 probe during the MRSA recognition process. (b) Fluorescence intensity of the HP during the target recognition mediated proximity ligation process. (c) Fluorescence intensity of the method when essential experimental components existed or not. ***, P ＜0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4505973/v1/e80f9c1071486afb22979b8f.png\"},{\"id\":58335451,\"identity\":\"4146bea0-1cb5-4291-9feb-733b41b91be3\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 05:20:15\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":111077,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe optimization of experimental conditions.\\u003c/strong\\u003e (a) F/F\\u003csub\\u003e0\\u003c/sub\\u003e (F and F\\u003csub\\u003e0\\u003c/sub\\u003e represent fluorescence intensity of the reaction mixture at 490 nm with and without existence 5000 cfu/mL of MRSA) of the method with different HP concentration. (b) F/F\\u003csub\\u003e0\\u003c/sub\\u003e of the method with different Bst polymerase concentration (U/μL). (c) F/F\\u003csub\\u003e0\\u003c/sub\\u003e of the method with different reaction time (min). (d) F/F\\u003csub\\u003e0\\u003c/sub\\u003e of the method with different concentration of ThT (mM).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4505973/v1/9f066b471fae321f8b6a873b.png\"},{\"id\":58335450,\"identity\":\"42904142-510d-4755-bc84-c78a6c3345b5\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 05:20:15\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":291053,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnalytical performance of the method for MRSA detection.\\u003c/strong\\u003e (a) Fluorescence spectrum of the method when detecting different concentrations of MRSA. (b) Fluorescence intensities at 480 nM of the method when detecting different concentrations of MRSA. (c) Linear correlation between the fluorescence intensities at 480 nM of the method and the logarithmic concentrations of MRSA. (d) Fluorescence intensities of the method when detecting different bacteria. (e) Fluorescence value recorded from the microwell plate when detecting different concentrations of various bacteria and MRSA. ***, P ＜0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4505973/v1/9ccd80aa39ec5befe1d62a79.png\"},{\"id\":58335449,\"identity\":\"d98aad11-0e06-4cc3-b507-a7dd349b68c1\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 05:20:15\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":125617,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eClinical application of the method for MRSA detection.\\u003c/strong\\u003e (a) MRSA concentration by the method and by colony counting method. (b) Fluorescence value recorded from the microwell plate when detecting different concentrations of MRSA (cfu/mL).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4505973/v1/98a8a00a703a16055849ce35.png\"},{\"id\":72776560,\"identity\":\"5b033461-9f6d-4971-bb4f-ce170dbd76b0\",\"added_by\":\"auto\",\"created_at\":\"2025-01-02 04:46:51\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1228964,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4505973/v1/74f5fbd2-ef9a-461f-908d-7632ba11fd86.pdf\"},{\"id\":58335447,\"identity\":\"d97b1fcf-3492-4759-bc7b-f8a1ad9cbd42\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 05:20:15\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":13322,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supportinginformation.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4505973/v1/1f7eeb71f93f5087644b7c6a.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Proximity Ligation Mediated Signal Recycling with Primer Exchange Reaction for Sensitive and Accurate Methicillin-Resistant Staphylococcus Aureus (MRSA) Detection\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eThe prevailing idea that the majority of surgical site infections (SSIs) that arise after elective surgery for liver cancer, following normal methods of antisepsis, are caused by intraoperative contamination, has not been substantiated [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Bacterial infectious diseases have emerged as a significant worldwide problem, posing a serious threat to human health [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Moreover, increasing evidence suggests that bacteria can also indirectly contribute to the development and advancement of other diseases, including cancer. There has been a rise in drug-resistant bacteria in recent years, resulting in a significant increase in the challenge and expense of developing new effective antibacterial therapeutic strategies. Methicillin-Resistant \\u003cem\\u003eStaphylococcus Aureus\\u003c/em\\u003e (MRSA) is a prominent contributor to intractable bacterial infections [\\u003cspan additionalcitationids=\\\"CR5\\\" citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. Hence, the accurate and highly sensitive identification of MRSA from clinical samples is crucial for the early detection and treatment of MRSA related infection. The \\u003cem\\u003emecA\\u003c/em\\u003e gene mediates methicillin resistance by expressing penicillin binding proteins (PBP) and PBP2a [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. Therefore, the detection of the \\u003cem\\u003emecA\\u003c/em\\u003e gene or its product PBP2a is frequently used as an additional biomarker to quickly confirm methicillin resistance.\\u003c/p\\u003e \\u003cp\\u003eSeveral MRSA detection approaches have been developed in recent years, primarily categorized into \\u003cem\\u003emecA\\u003c/em\\u003e gene sequencing [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e] and membrane protein analysis methods [\\u003cspan additionalcitationids=\\\"CR11 CR12\\\" citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. Despite the reliability and accuracy of the procedure, there are still significant deficiencies in these methods. Conventional colony counting techniques, which are commonly employed in clinical laboratories, are criticized by complicated procedures and inability to precisely identify colonies during the first phase of disease and assess treatment resistance [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. DNA sensors are highly regarded for the exceptional detection sensitivity; however, they often face criticism due to the possibility of false positive results caused by interfering components present in clinical samples [\\u003cspan additionalcitationids=\\\"CR16\\\" citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Hence, there is a significant promise in promoting a precise and highly sensitive technique for detecting MRSA.\\u003c/p\\u003e \\u003cp\\u003eGiven that MRSA has the ability to express several types of proteins such as PBP2a and protein A, multiple analytical techniques have been published to measure MRSA levels by identifying a MRSA specific protein. However, proteins released by the cell lysis and MRSA may generate undesired inaccurate signals. To reduce interference, two or more recognition sites are needed simultaneously. The recently developed dual-recognition [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e] mediated MRSA identification strategy has demonstrated promising advancements in detection accuracy, reliability, and low background noise. This strategy involves the simultaneous targeting of two membrane proteins using dual aptamers [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e], dual antibodies [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e], or a combination of aptamer and antibody [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e], as well as aptamer and cholesterol [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Although significant advancements have been made, these techniques may still encounter challenges, such as multiple separation and limited signal amplification efficiency. Therefore, there is still a significant need for MRSA detection methods that are simple, sensitive, and easy-to-operate. The proximity hybridization approach, reliant on simultaneous recognition of the target molecules by both DNA probes, has demonstrated exceptional selectivity. Furthermore, the proximity hybridization product can be additionally integrated with a conventional nucleic acid amplification technique to enhance sensitivity.\\u003c/p\\u003e \\u003cp\\u003eWe assume that the issues may be resolved by combining dual antibody-based target recognition with proximity ligation-mediated signal recycling (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). This approach specifically targets protein A, a membrane protein that is present in both MRSA, and PBP2a. The PBP2a-specific antibody and protein A-specific antibody are each bound to two distinct nucleic acid sequences to create the probes for proximity ligation (P1 and P2). The P1 and P2 probes consist of four functional sections: \\u0026ldquo;Antibody\\u0026rdquo; for target recognition, \\u0026ldquo;Spacer\\u0026rdquo;, \\u0026ldquo;Complementary\\u0026rdquo;, and \\u0026ldquo;Trigger\\u0026rdquo;. In the presence of MRSA, the P1 and P2 probes specifically attach to the protein A and PBP2a on the surface of MRSA, respectively. This enables the close proximity of the \\u0026ldquo;C1\\u0026rdquo; and \\u0026ldquo;C2\\u0026rdquo; sections, allowing for proximity hybridization. The close connection between the P1 and P2 probes results in the formation of an intact \\u0026ldquo;Trigger sequence\\u0026rdquo;, which can be used to enhance the following signal. Specifically, the \\u0026ldquo;Trigger sequence\\u0026rdquo; unfolds the hairpin probe (HP) to release the toehold portion. The toehold region forms a binding interaction with the \\u0026ldquo;2\\u0026rdquo; region of the primer exchange reaction probe (PER probe). With the aid of DNA polymerase, the chains are elongated using the \\u0026ldquo;toehold\\u0026rdquo; part and \\u0026ldquo;2\\u0026rdquo; section as primers. Consequently, the \\u0026ldquo;Trigger sequence\\u0026rdquo; is released, leading to the formation of a signal cycle. Additionally, a single-stranded DNA (ssDNA) chain containing the transcribed \\u0026ldquo;3*\\u0026rdquo; section is appended to the end of the \\u0026ldquo;toehold\\u0026rdquo; region of HP. The nicking site enzyme recognizes the specific sequence \\u0026ldquo;3*\\u0026rdquo; and creates a nicking site. The elongation of the ssDNA chain is halted by the \\u0026ldquo;Stop site\\u0026rdquo; in the PER probe and competed by the \\u0026ldquo;1\\u0026rdquo; segment. Consequently, a substantial quantity of \\u0026ldquo;4*\\u0026rdquo; sequences are generated, specifically G-quadruplexes. A multitude of G-quadruplex sequences interact with thioflavin T (ThT) to generate substantial fluorescence, enabling the detection of MRSA with excellent sensitivity and precision.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"2. Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Chemicals and materials\\u003c/h2\\u003e \\u003cp\\u003eMRSA strain (ATCC 17802), \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e (ATCC 25922), \\u003cem\\u003ePseudomonas aeruginosa\\u003c/em\\u003e (ATCC 19115), and \\u003cem\\u003eStreptococcus pneumonia\\u003c/em\\u003e (ATCC 50761) were provided by the American Type Culture Collection (ATCC). The design of all oligonucleotides was based on previous studies (\\u003cb\\u003eTable \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e\\u003c/b\\u003e) and they were produced by Shanghai Sangon (Shanghai, China). All of the oligonucleotides were utilized without additional purification. Nb.BbvCI, Bst polymerase, and deoxy-ribonucleoside triphosphate (dNTPs) were obtained from New England Biolabs, Inc. (Beijing, China). Thioflavin T (3,6-dimethyl-2-(4-dimethylaminophenyl)benzo-thiazolium cation, ThT) were obtained from Sigma-Aldrich (Shanghai, China). Diethypyrocarbonate (DEPC)-treated water (DNase, RNase free) obtained from Beyotime Institute of Biotechnology (Shanghai, China) was used in all experiments. More experimental details were shown in electronic supporting information (ESI).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Bacteria culture\\u003c/h2\\u003e \\u003cp\\u003eThe MRSA bacteria were cultured at a temperature of 37\\u0026deg;C for a period of 18 hours using a solution containing 3% NaCl. \\u003cem\\u003eE. Coli\\u003c/em\\u003e and \\u003cem\\u003eP. aeruginosa\\u003c/em\\u003e were cultivated in Lysogeny Broth (LB) medium for 12 hours at a temperature of 37\\u0026deg;C with a rotation speed of 200 rpm. The MRSA bacteria were grown in a 3% NaCl alkaline protein aqueous medium for 18 hours for the CFU test. The cell suspension was serially diluted, followed by 50 \\u0026micro;L of each gradient plated in triplicate on a TCBS dish and incubated at 37\\u0026deg;C for 24 h. Colonies were then counted and the average of three parallel dishes was recorded.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Analytical procedure\\u003c/h2\\u003e \\u003cp\\u003eThe bacteria, at different concentrations, were incubated for 40 min in 200 \\u0026micro;L of binding buffer containing 0.5 \\u0026micro;M P2 probe and 1 \\u0026micro;M P1 probe, respectively. Subsequently, a hairpin molecule with a concentration of 150 nM and a PER probe with a concentration of 200 nM were introduced into the solution. Subsequently, the resultant combination was held at a temperature of 80\\u0026deg;C for a duration of 20 min and then cooled down to 25\\u0026deg;C. Subsequently, the combination was incubated with the ThT dye (10 mM) for an additional 30 min and then diluted in Tris-HCl solution to a volume of 200 \\u0026micro;L in order to proceed with fluorescence recording. The RF-6000 Shimadzu fluorescence spectrophotometer (Tokyo, Japan) was used to record the fluorescence spectra.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 Statistical data analysis\\u003c/h2\\u003e \\u003cp\\u003eEach test was repeated at least three independent replicates, which were displayed as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation (SD). Data were visualized using software GraphPad Prism 8.0 (CA, USA). The Student's t-test was used to analyze comparisons between two groups. Differences were considered significant at values of P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results and discussion\",\"content\":\"\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1 Feasibility characterization of the method\\u003c/h2\\u003e \\u003cp\\u003eTo validate the detection of MRSA using P1 and P2 probes, we labeled the ends of P1 and P2 probes with fluorescent dyes FAM and Cy3, respectively. The P1 and P2 probes were individually exposed to MRSA for a period of 30 min. Subsequently, any probes that did not bind to the MRSA were removed using centrifugation. The fluorescence signal of the sediment is detected and then compared. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea exhibits a significant augmentation in the fluorescence signals of FAM and Cy3 when MRSA is present, demonstrating the successful attachment of P1 and P2 probes to MRSA.\\u003c/p\\u003e \\u003cp\\u003eIn order to verify the disassociation of HP using proximity hybridization of P1 and P2 probes, FAM and BHQ are attached to both ends of HP. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb shows that the FAM fluorescence signal was significantly reduced when linear HP was folded into a hairpin structure, as a result of the quenching action of BHQ on the FAM signal. When P1 and P2 are present, the fluorescence intensity of HP remains low, indicating that HP is not activated in these circumstances. The fluorescence signal of FAM was significantly enhanced only when MRSA, P1, and P2 were all present simultaneously, indicating the unfolding of the HP hairpin structure. When both MRSA and P1 or MRSA and P2 were simultaneously present, the FAM signals consistently remained at a low level, indicating that the activation of HP need close proximity hybridization between P1 and P2 probe.\\u003c/p\\u003e \\u003cp\\u003eMultiple samples were prepared using different parameters to evaluate the effectiveness of the proximity hybridization-dependent approach for accurately detecting MRSA. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec illustrates that the blank sample shows a diminished level of fluorescence intensity due to the absence of G-quadruplex formation (column 1). Sample 2, which does not include the P1 probe (column 2) and P2 probe (column 3), has a minimal degree of fluorescence, indicating the absence of proximity hybridization in the absence of the probes. Sample 3 (column 4) shows a diminished fluorescence intensity because MRSA is not present, which prevents the occurrence of proximity hybridization-dependent PER response. The fluorescence signal of the sample was much increased when all essential components were included in the sensor system (column 5).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2 The optimization of experimental conditions\\u003c/h2\\u003e \\u003cp\\u003eOnce the potential of our signal amplification bio-sensing method for detecting MRSA was confirmed, we proceeded to optimize the experimental parameters that influenced the performance of this MRSA detection method. These parameters included the concentration of the HP, the duration of the enzyme reaction, the amount of Bst polymerase, and the concentration of ThT. The optimization findings are displayed in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, where F and F\\u003csub\\u003e0\\u003c/sub\\u003e represent the fluorescence intensity of the reaction mixture at 490 nm with and without the presence of 5 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e cfu/mL of MRSA, respectively. Initially, the HP concentration was examined. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, the fluorescence response steadily increases and reaches a plateau at a concentration of 100 nM of HP. Consequently, it was discovered that a concentration of 100 nM of HP is ideal for MRSA detection. Following the optimization of HP concentration, the dosage of Bst polymerase was measured ranging from 3 U/\\u0026micro;L to 18 U/\\u0026micro;L, with an increase of 3 U/\\u0026micro;L. The findings can be seen in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb. The fluorescence intensity rises as the amount of Bst polymerase grows and reaches its highest value at 15 U/\\u0026micro;L. Consequently, the concentration of Bst polymerase in following tests was 15 U/\\u0026micro;L. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec illustrates the correlation between the reaction time of the enzyme and the fluorescence response. The fluorescence response increases significantly within the time frame of 30 min to 120 min, however there is no noticeable change after 120 min. Furthermore, the impact of ThT at various doses on the fluorescence emission intensity of the reaction solution was investigated (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed). Within the range of concentrations examined, the fluorescence intensity increases until it reaches its highest value at 8 mM when the ThT concentration is raised. This indicates that the ideal concentration of ThT is 8 mM.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3 Detection of MRSA using the established strategy\\u003c/h2\\u003e \\u003cp\\u003eThe proposed technique was utilized for MRSA detection under optimum conditions, taking advantage of its strong potential to produce amplified signal outputs. Initially, we examined its viability. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea demonstrates that the fluorescence of the approach was insignificant when MRSA was not present. When MRSA was present at diverse concentrations (ranging from 10 to 10\\u003csup\\u003e6\\u003c/sup\\u003e cfu/mL), the fluorescence exhibited a significant increase as the MRSA concentration increased. Furthermore, we observed a direct relationship between the measured fluorescence intensity and the logarithm of MRSA concentration (C) within the range of 10 to 10\\u003csup\\u003e6\\u003c/sup\\u003e cfu/mL (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb), which covers a span of six orders of magnitude. The calibration equation can be expressed as: F\\u0026thinsp;=\\u0026thinsp;93.64*lgC\\u0026thinsp;+\\u0026thinsp;2.940. The correlation coefficient (R\\u003csup\\u003e2\\u003c/sup\\u003e) for this equation is 0.9869, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec. The exceptional sensitivity is mostly attributed to the simultaneous amplification of signals through both target recycling and PER process.\\u003c/p\\u003e \\u003cp\\u003eAn important obstacle in MRSA analysis is the capacity to precisely distinguish MRSA from a wide range of microorganisms. In this study, the specificity was evaluated using the detection of \\u003cem\\u003eE. Coli\\u003c/em\\u003e, \\u003cem\\u003eP. aeruginosa\\u003c/em\\u003e, and \\u003cem\\u003eS. pneumoniae\\u003c/em\\u003e. The proposed method allows for the detection of these interfering bacteria and their combination. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed demonstrates the contrast in fluorescence signals\\u0026rsquo; reaction to various bacteria. The signal was feeble in the presence of \\u003cem\\u003eE. Coli\\u003c/em\\u003e, \\u003cem\\u003eP. aeruginosa\\u003c/em\\u003e, and \\u003cem\\u003eS. pneumoniae\\u003c/em\\u003e, as opposed to MRSA. In addition, we also utilized the technique to identify a combination of bacteria (\\u003cem\\u003eE. coli\\u003c/em\\u003e, \\u003cem\\u003eP. aeruginosa\\u003c/em\\u003e, \\u003cem\\u003eS. pneumoniae\\u003c/em\\u003e, and MRSA), resulting in a substantial increase in fluorescence compared to the control group. Presently, point-of-care (POCT) is garnering growing interest in the detection of disease-related targets due to the simple detection procedure and devices. In this process, we combine all the elements of the approach and place them in a microwell plate to create a portable device for detecting MRSA. Subsequently, the microwell plate was employed to identify \\u003cem\\u003eE. coli\\u003c/em\\u003e, \\u003cem\\u003eP. aeruginosa\\u003c/em\\u003e, and \\u003cem\\u003eS. pneumoniae\\u003c/em\\u003e. The fluorescence intensity of these interfering bacteria, MRSA, and their combination was measured using a microplate reader. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee demonstrates a comparable fluorescence intensity trend, suggesting that the approach has significant promise for point-of-care testing (POCT) applications.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4 Analysis of MRSA from clinical samples\\u003c/h2\\u003e \\u003cp\\u003eInterference in the clinical samples can impede the binding of antibodies to the target protein and disrupt the proximity ligation process. Hence, it was imperative to evaluate the suitability of this approach for clinical sample detection. To replicate the clinical condition, MRSA with varying concentration gradients were introduced into serum. Serum samples were acquired by centrifuging a clinically obtained normal human blood sample. Initially, the amount of MRSA in clinical samples was determined using the conventional colony counting method and the suggested technique (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea). The results obtained from the four clinical samples using the method was in high agreement with those obtained using the traditional colony counting method, demonstrating the reliability of our suggested method. In order to showcase the possible POCT application of the proposed approach, we conducted a test on four clinical samples using a microplate under the optimized experimental conditions. The outcome, as depicted in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb, can specifically distinguish the interfering bacteria from samples, indicating the POCT application potential of the method.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Conclusion\",\"content\":\"\\u003cp\\u003eEfficiently and precisely identifying disease-causing bacteria in clinical samples is a global obstacle. In addition to the conventional colony count approach, two primary methods have been documented for the detection of bacteria: aptamer-based detection method and protein detection method. Generally, aptamer-based detection methods have been found to be more sensitive because aptamers may convert the levels of membrane protein into nucleic acid detection. On the other hand, protein detection methods assure high specificity by relying on the interaction between antigen and antibody. Nevertheless, the current methods for detecting MRSA still encounter the following issues: i) The existing MRSA detection methods lack enough sensitivity for trace amount MRSA detection; ii) The current MRSA detection technologies is not suitable for POCT applications; iii) The detection outcome is highly susceptible to interference from a single protein on the surface of MRSA. In this study, we present a novel method for detecting MRSA by combining the benefits of proximity ligation and PER procedure. We devised a sophisticated approach to concurrently identify the presence of protein A and PBP2a. When combined with a dual signal amplification, our findings demonstrate that MRSA can be accurately and directly detected with a high level of sensitivity. By implementing the proposed method in POCT, we anticipate its versatility in detecting various bacteria, with exceptional specificity and quick amplification.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eSupplementary Information\\u003c/strong\\u003e The online version contains supplementary material available at: xxx-xxx-xx.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e We appreciate the financial support from the Jiaozuo People\\u0026rsquo;s Hospital. We also thank the director of central laboratory for providing essential equipment.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe study was conceived by Huali Xu. Huali Xu conducted the lab work and wrote the manuscript. Xiangke Yang, Wen Wang, and Xiaomin Yuan assisted data analysis. All authors discussed and aided in interpreting the results.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability\\u003c/strong\\u003e All data generated and analyzed during this study are included in this article.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical Approval\\u003c/strong\\u003e This article does not contain any studies with human participants or animals performed by any of the authors.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent to Participate\\u003c/strong\\u003e Not applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for Publication\\u003c/strong\\u003e Not applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of Interest\\u003c/strong\\u003e The authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFund:\\u0026nbsp;\\u003c/strong\\u003eNot available\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eSeidelman J, Anderson DJ: Surgical Site Infections. \\u003cem\\u003eInfect Dis Clin North Am \\u003c/em\\u003e2021, 35(4):901-929.\\u003c/li\\u003e\\n\\u003cli\\u003eSeidelman JL, Mantyh CR, Anderson DJ: Surgical Site Infection Prevention: A Review. \\u003cem\\u003eJAMA \\u003c/em\\u003e2023, 329(3):244-252.\\u003c/li\\u003e\\n\\u003cli\\u003eLiu C, Bayer A, Cosgrove SE, Daum RS, Fridkin SK, Gorwitz RJ, Kaplan SL, Karchmer AW, Levine DP, Murray BE\\u003cem\\u003e et al\\u003c/em\\u003e: Clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children. \\u003cem\\u003eClin Infect Dis \\u003c/em\\u003e2011, 52(3):e18-55.\\u003c/li\\u003e\\n\\u003cli\\u003eBurnham CD, Leeds J, Nordmann P, O\\u0026apos;Grady J, Patel J: Diagnosing antimicrobial resistance. \\u003cem\\u003eNat Rev 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\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Methicillin-Resistant Staphylococcus Aureus (MRSA), G-quadruplex, primer exchange reaction, thioflavin T, proximity ligation assay\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4505973/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4505973/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eInfectious diseases have emerged as a significant global concern, posing a substantial burden in terms of the high morbidity and mortality, and presenting considerable challenges in clinical diagnosis and treatment. Therefore, it is highly-desired to develop new strategies for sensitive and accurate bacteria detection to address the global epidemic of antibiotic resistance.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eIn this study, a new technique combining proximity ligation and primer exchange reaction (PER) was developed for precise identification and highly sensitive detection of Methicillin-Resistant \\u003cem\\u003eStaphylococcus Aureus\\u003c/em\\u003e (MRSA). The antibodies recognize both protein A and PBP2a on the surface of MRSA, leading to the initiation of proximity ligation and PER process. The PER procedure generated a substantial number of G-quadruplex sequences, which subsequently bind with thioflavin T (ThT) to significantly amplify its fluorescence, enabling the detection of MRSA with a low detection limit of 3.5 cfu/mL.\\u003c/p\\u003e\\u003ch2\\u003eConclusion\\u003c/h2\\u003e \\u003cp\\u003eDue to its non-label format, high selectivity, and sensitivity, this method can serve as a practical and versatile approach for detecting different bacteria in the early stages of infectious diseases.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Proximity Ligation Mediated Signal Recycling with Primer Exchange Reaction for Sensitive and Accurate Methicillin-Resistant Staphylococcus Aureus (MRSA) Detection\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-06-14 05:20:10\",\"doi\":\"10.21203/rs.3.rs-4505973/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"51282fc1-2113-4f00-bd7f-40bbf8d49567\",\"owner\":[],\"postedDate\":\"June 14th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-01-02T04:38:44+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-06-14 05:20:10\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4505973\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4505973\",\"identity\":\"rs-4505973\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}