Visual detection for environmental antibiotic resistance genes by recombinase polymerase amplification combined with lateral flow dipstick

Visual detection for environmental antibiotic resistance genes by recombinase polymerase amplification combined with lateral flow dipstick | 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 Visual detection for environmental antibiotic resistance genes by recombinase polymerase amplification combined with lateral flow dipstick Yunqian Zhou, Fengming Wang, Jingyi Jiang, Ying Zhao, Qiang Du, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7655921/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The emergence of the antibiotic resistance genes(ARGs) has posed a significant challenge in controlling the spread of multidrug-resistant bacteria both in clinic and environment.To establish the purpose of detecting the environmental ARGs in room temperature and achieving visualization, this study developed a rapid detection by combining recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) targeting crucial ARGs including bla NDM , bla KPC , mcr , tetX1 and tetX2. Various primers and nfo probes were designed and LFD-RPA method was established, followed by the sensitivity, specificity and repeatability tests.Sensitivity was evaluated with ten-fold serial dilutions of plasmid standards, specificity was tested against environmental isolates lacking target genes, and reproducibility was assessed in ten replicates. Results showed that the limits of detection ranged from 1.061×10 1 to 1.803×10 2 copies/mL,with tetX1 demonstrating the highest sensitivity at 1.061×10 1 copies/mL .The LFD-RPA system showed 100% specificity without cross-reactivity, and exhibited great repeatability. Thus, the LFD-RPA platform provided a highly sensitive, specific and one-site detection for real-time environmental monitoring of key ARGs. Antibiotic resistance gene Recombinase polymerase amplification Lateral flow dipstick point-of-care testing Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The intensive dependence on antimicrobial agents has caused the prevalence of antimicrobial resistance (AMR) become increasingly critical, with microorganisms frequently harboring multiple antimicrobial resistance genes (ARGs)[1, 2]. Antibiotics are utilized not only in clinical therapeutics but also extensively in veterinary medicine and aquaculture, resulting in widespread environmental dissemination of residual compounds[3]. Low-level antibiotic exposure induces microbial resistance and facilitates the horizontal transfer of resistance genes via mobile genetic elements across species, thereby accelerating the dissemination of antimicrobial resistance[4, 5].In 2024, The World Health Organization has identified carbapenem-resistant Enterobacterales and Acinetobacter baumannii as the most problematic multidrug-resistant bacteria [6]. Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM) are common carbapenemases encoded by the bla KPC and bla NDM genes, respectively[7], and are capable of clonal transmission between humans and the environment[8].The increasing incidence of carbapenem-resistant bacteria and the paucity of novel antimicrobials have heightened dependence on colistin as a last-resort antibiotic, thereby driving the emergence of the colistin resistance[9]. And the coexistence of carbapenemase genes and mobile colistin resistance (mcr) genes in the same bacterial isolates can lead to multidrug resistance(MDR)[10].Moreover, the tigecycline served as the final line of defense against severe infections by pan-drug-resistant bacterial pathogens has been challenged by the recent emergence of the mobile tet(X) orthologs that can confer tigecycline resistance [11]. Therefore, carbapenem antibiotics exhibit widespread resistance, with recent emergence of colistin and tigecycline resistance, emphasizing the urgent need for rapid detection methods to improve antimicrobial resistance surveillance and clinical intervention. Conventional antimicrobial susceptibility testing (AST) methods, such as disk diffusion, agar dilution and broth microdilution are limited by phenotypic screening and prolonged detection cycles[12], whereas genotypic detection methods offer higher sensitivity and specificity, significantly reducing the time for resistance gene detection[13]. However, most genotyping detection methods depend on thermal cycling nucleic acid amplification requiring specialized laboratory equipment, limiting their applicability in point-of-care testing (POCT). To overcome this limitation, isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), and recombinase polymerase amplification (RPA) , have been developmented[14]. RPA, acknowledged as a viable alternative to polymerase chain reaction (PCR) for nucleic acid detection, is an innovative isothermal amplification technique that emulates the intracellular nucleic acid replication process, leveraging the replication mechanism of T4 bacteriophage[15].This technique relies on three key enzymes: a recombinase that binds single-stranded nucleic acids, single-strand DNA-binding protein (SSB), and strand-displacing DNA polymerase, all of which remain active at ambient temperature, with optimal reaction conditions between 37°C and 42°C. RPA amplification reactions have been demonstrated to achieve exponential growth of target genes in a very short time. RPA technology, when used with fluorescent probes, can monitor amplification in real time. It can also be integrated with lateral flow strips, simplifying workflows and reducing costs, making it suitable for POCT. In this study, a visualisation method for ambient temperature amplification targeting five antibiotic resistance genes ( bla NDM , bla KPC , mcr , tetX1 , and tetX2 ) was established by combining RPA and lateral flow dipstick (LFD). This method provides a novel approach and methodology for the rapid detection of antibiotic resistance genes in environmental microorganisms. 2. Materials and Methods 2.1 Bacterial Strain Collection and Identification A total of 339 bacterial isolates were obtained from environmental samples collected within local hospitals in Changzhou from 2012 to 2021. The sampling, collection, and microbiological analysis procedures were conducted in accordance with the standards outlined in GB15982 "Hygienic standard for disinfection in hospitals" (2012).All strains were identifided utilizing a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) system (VITEK-MS, BioMérieux, France). 2.2 Phenotypic and genotypic antimicrobial resistance of strains We selected Klebsiella pneumoniae and Escherichia coli isolates for subsequent microbiological characterization.The reference method for AST was the microdilution method standardized by “Clinical and Laboratory Standards Institute”(CLSI)[16],utilizing an automated antimicrobial susceptibility platform(Thermo Fisher Scientific, USA) . The antibiotics evaluated included ampicillin, piperacillin, cefuroxime, ceftriaxone, cefepime, levofloxacin, imipenem, meropenem, ertapenem, polymyxin, and tigecycline. Subsequently, the multi-drug-resistant strains of K. pneumoniae and E. coli, which demonstrated resistance phenotypes, were selected as the subjects for the study. The bla NDM , bla KPC , mcr, tetX1, and tetX2 genes carried by multi-drug resistant K. pneumoniae and E. coli were detected by PCR, with amplicon visualization conducted through 1% agarose gel electrophoresis. The products were sent to Genewiz for Sanger sequencing, and the obtained gene sequences were validated for sequence fidelity through BLAST analysis against the GenBank database. 2.3 Primer and probe design Conserved regions from 22 bla NDM genotypes, including bla NDM-1 , bla NDM-2 , bla NDM-3 , bla NDM-4 , bla NDM-5 ,and bla NDM-6 (GenBank accession numbers KX999121.1, KU510393.1, JQ734687.1, KP772213.1, KP772211.1, NG049338.1, etc.), were selected through sequence alignment as the template for the RPA of the bla NDM gene. Addtionally, conserved sequences from various genotypes of the bla KPC , mcr , tetX1 , and tetX2 genes were also employed as templates for RPA. The amplified target sequences were cloned into the pUC-18 plasmid vector to generate specific recombinant constructs. The PCR primer sequences used for plasmid construction are listed in Table 1. In this study, 10 sets of RPA primers were designed to target the bla NDM , bla KPC , mcr , tetX1 , and tetX2 genes, along with nfo fluorescent probes for the LFD-RPA, designated as A1, A2, B1, B2, C1, C2, D1, D2, E1, and E2. The RPA primer pairs are optimized to a length of 30-35 bp, with the 5’ end of the reverse primers labeled with biotin. The nfo probes are 48-55 bp, and a tetrahydrofuran (THF) group replaces the adenylate site as the recognition site of endonuclease, located at least 30 bp away from the 5′ end and 15 bp away from the 3’ end. Moreover,the 5' end of the probe is labeled with a FAM fluorophore, while the 3’end is modified with a C3 spacer to prevent extension. Both primers and probes were synthesized by Genewiz, as detailed in Table 2. Table 1 PCR Primer Table for bla NDM 、 blaKPC , mcr , tetX1 and tetX2 genes Name Sequence (5’-3’) products(bp) bla NDM F CATTAGCCGCTGCATTGA 400 bla NDM R GCAAGCTGGTTCGACAACGC bla KPC F CATTCAAGGGCTTTCTTGCTGC 538 bla KPC R ACGACGGCATAGTCATTTGC mcr F CGGTCAGTCCGTTTGTTC 309 mcr R CTTGGTCGGTCTGTAGGG tetX1 F CGAAAAATGTTGCTTGGCAGCTT 486 tetX1 R AGTTGTTGAACGAATTAACTCC tetX2 F CGGGATGTCCAAGGTAAGAAAA 343 tetX2 R TGACAACGTCGTATGAATCAA Table 2 The Primer and Probe Sequences for LFD-RPA Name Primer direction Sequence(5’-3’) bla NDM A1 Forward CTTATGCCAATGCGTTGTCGAACCAGCTTGCCC Reverse Biotin-CCCAACGGTGATATTGTCACTGGTGTGGCC probe 5`6-FAM-CAACACAGCCTGACTTTCGCCGCCAATGGCTG/idSp/GTCGAACCAGCAACCGCG-3`C3-Spacer bla NDM A2 Forward CATTAGCCGCTGCATTGATGCTGAGCGGGTGCATGCCC Reverse Biotin-CCCTGACGATCAAACCGTTGGAAGCGACTGCCC probe 5`6-FAM-AGCTCGCACCGAATGTCTGGCAGCACACTTCC/idSp/ATCTCGACATGCCG-3`C3-Spacer bla KPC B1 Forward TGCCACCGCGCTGACCAACCTCGTCGCGGAAC Reverse Biotin-AAGCCCTTGAATGAGCTGCACAGTGGGAAGCG probe 5`6-FAM-GACTTTGGCGGCTCCATCGGTGTGTACGCGATGGA/idSp/ACCGGCTCAGGCGCAACTGTAAGTTA-3`C3-Spacer bla KPC B2 Forward AGGAGCGCTTCCCACTGTGCAGCTCATTCAAGGGCT Reverse Biotin-TCATGCCTGTTGTCAGATATTTTTCCGAGATG probe 5`6-FAM-TGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGC/idSp/GGACACACCCATCCGTTACGGCA-3`C3-Spacer mcr C1 Forward CTAAAGCCTGTGTTGATTTTGCTATTAATCATGGG Reverse Biotin-CCCAATCGGCGCATCAAACCCTTGCCCCAA probe 5`6-FAM-CGGTCTATGATACGACCATGCTCCAAAATGC/idSp/CTACAGACCGACCAAGCCGAG-3`C3-Spacer mcr C2 Forward AGACGCGGTACAAGCAACCAAGCCTGATATGCG Reverse Biotin-TGGTCACGCCATCGATCTTGGCAAGCTGTGG probe 5`6-FAM-CGTCGTCGGTGAGACGGCACGCGCCGATCATG/idSp/TCAGCTTCAATGGCTATGA-3`C3-Spacer tetX1 D1 Forward TTAAAAGGTTACTCCTATTTGGGATTATAATA Reverse Biotin-TATGGAGCCAATAAGACCAAACGCTTTACC probe 5`6-FAM-CTGGCTCGATTTATTCAAGGAGCTGGTGCAGC/idSp/GCATTTCCAGCACTCGTG-3`C3-Spacer tetX1 D2 Forward TTATTGGCTCCATAGTAGCTATGGGAGAAG Reverse Biotin-AGTATAATTCCTTTGATATCAAAATGACCTT probe 5`6-FAM-TTCTACTCATTCCTATGATAACAATTATCACTGT/idSp/CCGTTTCTTATGAAATTATTAA-3`C3-Spacer tetX2 E1 Forward GCTTGCACTTTATGCGTTAATGCAGGTTATCT Reverse Biotin-CAAACGGCCTAAATACAGCATCCAAAGCGCACT probe 5`6-FAM-TCTGACCGATTTGGTCGGCGCCCAGTGCTGTTG/idSp/TGTCATTAATAGGCGCATCGCTG-3`C3-Spacer tetX2 E2 Forward CATGAGCAAGGTGCTTTACAGGGATTATTG Reverse Biotin-CTAAACCAATAATCCAAATCCAGCCATCCCA probe 5`6-FAM-GCAACCGGTGTTATTGGCCCATTACTGTTTAC/idSp/GTTATTTATAATCATTCAC-3`C3-Spacer 2.4 Establishment of the LFD-RPA reaction system Amplification assays targeting plasmid containing bla NDM , bla KPC , mcr , tetX1 , and tetX2 genes were conducted using the primer pairs and probes detailed in Table 2. The RPA reactions were carried out using the TwistAmp nfo kit (TwistDX, UK). The reaction mixture consisted 2.1 μl of the upstream primer (10 μM), 2.1 μl of the downstream primer (10 μM), 0.6 μl of the nfo probe (10 μM), 29.5 μl of Rehydration Buffer, and a total of 13.2 μl of DNA template and nuclease-free water, yielding a final volume of 47.5 μl. Immediately, 2.5 μl of 280 mM magnesium acetate was added into the system, and the reaction was incubated at 37–39°C for 20–40 minutes. Subsequently, the RPA reactions were performed on a real-time PCR system (QuantStudio 5, ABI) with an isothermal cycling protocol: an initial pre-denaturation at 40°C for 1 minute, followed by 40 cycles of amplification at 40°C for 31 seconds. Then, 5 μL of the amplification product was diluted into 95 μL of PBST to prepare the lateral flow immunoassay. The prepared mixture was applied to a lateral flow strip embedded with anti-FAM antibodies, using the HybriDetect - Universal Lateral Flow Assay Kit(Milenia Biotec,Germany), with results interpreted within 5 minutes. 2.5 Sensitivity of the LFD-RPA assay The recombinant plasmisds were converted to copy numbers by measuring the concentration of plasmids , following the formula: DNA copies/μL = (ng/μL × 6.02 × 10 23 × 10 −9 )/(Fragment length (bp)×660).Starting with a DNA template concentration of 40 ng/mL, the plasmids were serially diluted ten-fold. The prepared bla NDM ( 1.764×10 9 copies/mL-1.764×10 0 copies/mL), bla KPC (1.800×10 9 copies/mL-1.800×10 0 copies/mL), mcr (1.803×10 9 copies/mL-1.803×10 0 copies/mL), tetX1 (1.061×10 9 copies/mL-1.061×10 0 copies/mL), and tetX2 (1.271×10 9 copies/mL-1.271×10 0 copies/mL) genes were utilized for sensitivity testing, and different copy numbers of the genes were used as templates for LFD-RPA.The limit of detection (LOD) of the five genes were determined via observation of the color intensity of the dipsticks test line. 2.6 Specificity of the LFD-RPA Assay Phenotypic isolates exhibiting resistance to non-KPC, NDM, MCR, and TETX1/2 among K. pneumoniae, Pseudomonas aeruginosa, Shigella spp., Staphylococcus aureus, Sphingomonas paucimobilis, and Enterobacter cloacae were obtained through routine epidemiological surveillance conducted by Changzhou Disease Control and Prevention Centre. The strains were performed using LFD-RPA reactions separately, and the results were compared with those of the positive plasmid control groups to evaluate the specificity of the LFD-RPA system. 2.7 Reproducibility of the LFD-RPA assay We Selected the amplification systems with higher sensitivity from Table 2 and perform ten consecutive repetitions of LFD-RPA. The reproducibility of the LFD-RPA amplification is evaluated by analyzing the positive rate across these replicates. 2.8 Statistical analysis Grayscale image analysis of the test line on the lateral flow strip was conducted using ImageJ software to quantify the grayscale value. The results were expressed as the mean ± standard deviation (SD), and all statistical analyses were performed using the SPSS 26.0 package. 3. Results 3.1 Identification of bacterial strains and antimicrobial resistance genes Through identification using VITEK-MS and AST via microdilution, a total of 65 isolates of K. pneumoniae and E. coli strains carrying genes for carbapenems, tigecycline, and polymyxins were detected. Among these, three isolates harbored the bla NDM gene, eleven carried the bla KPC gene, one possessed the mcr gene, and one contained both the tetX1 and tetX2 genes. 3.2 Schematic of the LFD-RPA detection process The schematic workflow for the detection of ARGs is depicted in Figure 1. The extracted genomic DNA functions as the template for RPA amplification, with the nfo probe conjugated with FAM at the 5' end and the reverse primer labeled with biotin. Consequently, a positive RPA reaction yields dual-labeled amplicons that specifically bind to streptavidin immobilized on the Test line (T line), producing a visible signal. Simultaneously, the Control line (C line) also shows a colorimetric response. In the case of a negative result, the FAM-labeled probe is captured by anti-FAM antibodies, while the resulting nucleic acid fragments, lacking biotin, cannot bind to streptavidin, resulting in no visible band at the T line. 3.3 Sensitivity analysis of the LFD-RPA assay The sensitivity of the LFD-RPA was analyzed using various copies of recombinant plasmids containing the bla NDM , bla KPC , mcr , tetX1 , and tetX2 genes, all with an initial concentration of 40 ng/mL. The highest number of copies at which a positive result was observed was designated as the LOD. Lateral flow assay results are shown in Figure 2, with the bla NDM gene exhibiting a maximum detectable dilution of 10 8 , corresponding to the LOD of 1.764 ×10 1 copies/mL. Results for other genes are detailed in Table 3 and Figure 2. Except for the mcr gene, which had a higher detection limit of 1.803×10 2 copies/mL, the LOD of the nfo-RPA amplification for the other genes ranged between 10 and 20 copies/mL, with tetX1 demonstrating the highest sensitivity at 1.061×10 1 copies/mL. Figure 2B also showed that among the ten sets of primers and nfo-probes, the specific primer and probe sets that were categorized within groups A1, B1, C2, D2, and E2 demonstrated a significantly higher level of sensitivity, with grayscale value exhibiting a linear correlation with template concentration. Notably, the A1 set targeting bla KPC exhibited the minimal slope in the graysclae value versus dilution curve, indicating sustained high amplification efficiency at low template concentrations. These results further validated the ultra-high sensitivity and practical applicability of nfo-RPA for rapid on-site detection of resistance genes. Table 3 Detection limits of LFD-RPA in different amplification systems gene LOD（copise/ mL） maximum detectable dilution amplification systems bla NDM 1.764×10 1 10 8 A2 bla KPC 1.800×10 1 10 8 B1 mcr 1.803×10 2 10 7 C1 tetX1 1.061×10 1 10 8 D2 tetX2 1.271×10 1 10 8 E2 3.4 Specificity and reproducibility analysis of LFD-RPA assay Strains collected in the routine antimicrobial resistance surveillance work were subjected to the Specificity analysis for the LFD-RPA assays. The isolates included K. pneumoniae strains exhibiting resistance phenotypes absent of bla NDM , bla KPC , mcr , tetX1 , or tetX2 genes (resistance profile: AMP-ETP-CTX-CAZ-CIP positive, designated as T1), P.aeruginosa susceptible strains (T2), Shigella (SXT-AZM-E-CIP-AZT, T3), S. aureus (OXC-ERY-LEV-TET-GEN-VAN, T4), S. marcescens (C-CAZ, T5), and E. cloacae (CAZ-AZT-SAM-CZO-TZP, FEP, T6). These isolates, along with corresponding plasmid controls containing the target resistance genes (T7), went through the specificity validation across five distinct LFD-RPA platforms. The results presented in Figure 3 illustrated that the LFD-RPA assays targeting the bla NDM , bla KPC , mcr , tetX1 , and tetX2 genes demonstrated high specificity for their respective resistance phenotypes, with no false-positive amplifications observed. To evaluate the reproducibility of these assays, the most sensitive primer and probe combinations from ten selected assays were tested using ten-fold serial dilutions of the respective plasmid templates, with each assay performed in ten replicates. The findings, depicted in Figure 4, showed consistent positive detection across all replicates, confirming the robustness and reliability of the LFD-RPA methodology for resistance gene detection. 4. Discussion Currently, the rapid dissemination of antimicrobial-resistant microorganisms has emerged as one of the foremost global threats to human health. ARGs have transcended clinical boundaries, spreading extensively across aquatic systems, soils, the atmosphere, and the food chain[17]. Consequently, ARG-mediated environmental contamination has become a significant public health concern worldwide.Mobile genetic elements (MGEs) such as plasmids, integrons and transposons facilitate the horizontal transfer of critical resistance genes such as bla KPC , thereby promoting the dissemination of ARGs within bacterial communities and enhancing the spread of AMR[18].The proliferation of ARGs in environmental matrices affects ecological integrity and presents potential public health hazards via contaminated potable water, food sources, and aerosols[19].Therefore, the implementation of rapid, on-site monitoring of environmental antimicrobial resistance genes is of critical importance for the prevention and control of the dissemination of antibiotic resistance.This study focuses on five representative ARGs: bla NDM , bla KPC , mcr , tetX1 , and tetX2 develop a visual detection method based on LFD-RPA for ambient temperature application.Comprehensive validation encompassing sensitivity, specificity, and reproducibility analyses confirms that this methodology offers a practical and readily deployable platform for environmental surveillance of antimicrobial resistance. Bla NDM , bla KPC , mcr , tetX1 , and tetX2 represent the key resistance determinants for the three major last-resort antibiotic classes: carbapenems, polymyxins, and tigecycline. These genes play a critical role in clinical and environmental antimicrobial resistance studies, and their presence and spread pose a substantial threat to public health[20].For instance, the metallo-β-lactamases and serine carbapenemases encoded by the bla NDM and bla KPC genes can hydrolyze virtually all β-lactam antibiotics, resulting in bacterial pan-resistance[21, 22],The mcr gene family confers colistin resistance through the modification of lipid A , with colistin serving as the ultimate therapeutic choice for treating carbapenem-resistant Gram-negative bacterial infections[23].Enzymes encoded by tetX1 and tetX2 belong to the TetX enzyme family, which catalyzes the hydroxylation of tetracycline-class antibiotics (including tigecycline), resulting in their conversion to inactive metabolites and thereby mediating bacterial tigecycline resistance[24].The coexistence of these five genes within the same environmental micro-ecological niche may induce multidrug resistance and potentially disseminate to pathogenic bacteria via horizontal gene transfer, resulting in a clinical scenario where therapeutic options become ineffective[25].Therefore, implementing joint surveillance of these genes is crucial for assessing AMR risks in the environment and formulating subsequent targeted intervention strategies. PCR, multiplex PCR, and real-time quantitative PCR have been widely employed in the detection of ARGs[26, 27]. However, these conventional approaches rely on gel electrophoresis for result interpretation or necessitate specialized real-time fluorescence quantification instruments, which are not only time-consuming but also demand advanced laboratory equipment[28].To address this challenge, this study established a LFD-RPA system utilizing the nfo probe, to fulfill POCT requirements. RPA enables exponential amplification at 37–42°C without thermal cycling. By incorporating dual labeling with FAM-biotin into the amplification products via the nfo probe, RPA products are captured on a lateral flow strip by anti-FAM gold-labeled antibodies, forming a visually detectable Test line, which can be interpreted within 5 minutes[29]. Compared to traditional PCR, the LFD-RPA method eliminates the need for costly equipment, complex optical systems, and data analysis software,with the entire process can be performed at room temperature, offering a simple operation and shorter turnaround time Designing primers and probes is the most critical and challenging step in establishing RPA amplification. In this study, we meticulously designed ten primer-probe sets targeting five representative ARGs. Specifically, for the bla NDM gene, we considered 22 different variants, including bla NDM-1 , bla NDM-5 , and bla NDM-9 . A previous research has indicated that these three variants are the most prevalent among bla NDM familyand are widely distributed globally, particularly across Asia, Europe, and North America[30]. Consequently, the primers and probes designed for the bla NDM gene in this study exhibit both broad representativeness and applicability.The mcr-1 gene is globally recognized as the most widely reported plasmid-mediated colistin resistance gene ,and the mcr-3 gene has been frequently detected in Thailand, Vietnam, and China, particularly in Escherichia coli isolates from clinical and agricultural sources[31]. This study involved the development of two sets of primers and probes which precisely target conserved sequence from mcr-1 to mcr-10 , thereby ensuring comprehensive and accurate detection. The success of RPA amplification critically depends on the selection of conserved regions within the respective ARGs, as well as the sensitivity, reproducibility, and specificity of the primers and probes. Consequently, the primers and probes were subjected to rigorous experimental validation and optimization, as detailed below. Figure 2 illustrates the visualization of plasmid templates in LFD-RPA at 10-fold gradient dilutions. As the template concentration decreases, the color of the test line transitions gradually from deep red to a lighter shade. Nevertheless, it remains discernible by the naked eye even at the furthest right, highlighting the exceptional sensitivity of the system. Through grayscale scanning and subsequent conversion of copy numbers, LOD were determined as follows: tetX1 at 1.061×10 1 copies/mL, bla NDM at 1.764×10 1 copies/mL, bla KPC at 1.800×10 1 copies/mL, and tetX2 at 1.271×10 1 copies/mL. The LOD for mcr was slightly higher at 1.803×10 2 copies/mL, yet notably lower compared to the study of Gorecki et al. In their study, researchers utilized quantitative PCR to monitor mcr genes in the environment, detecting mcr-1 at 9.35×10 2 copies/mL and mcr-3 at 3.17×10 4 copies/mL[32].In the specificity experiment(Figure 3), six clinical isolates (T1-T6) lacking the target gene but possessing other drug resistance profiles, along with five positive plasmids (T7) containing the detection gene, were selected. Results revealed the absence of T-lines for T1-T6, with only colored C-lines observed. In contrast, T-lines for T7 were clearly visible, demonstrating the system's 100% specificity without cross-reaction to non-target genes. Fig. 4 showed that reproducibility tests were repeated 10 times with the same batch and the same operator, using 10 7 -fold diluted plasmid as template. The 10 T lines had the same depth, ImageJ gray scanning showed CV <5%, indicating that the system had excellent intra-batch repeatability. The remarkable characteristic of this study is that the established amplification method for detecting environmental resistance genes can be advanced and visible at room temperature without relying on complex large-scale instruments and equipment, thus effectively realizing the portability and miniaturization of detection equipment. This portable and compact design greatly meets the need for rapid response in environmental sample detection. Through this technical means, we have not only significantly improved the speed of testing, but also significantly reduced the economic cost of testing, making rapid on-site detection of antibiotic resistance genes feasible. 5. Conclusion To conclude , this study successfully designed primers and probes sets targeting five key ARGs including bla NDM , bla KPC , mcr , tetX1 and tetX2 , and developed a LFD-RPA system achieved visual detection at room temperature with sensitivity of 1.061×10 1 -1.803×10 2 copies/mL, specificity of 100% and reliable repeatability. It breaked through the dependence on instruments and laboratories, combined the requirements of high sensitivity and field ease of use, and provided a feasible new scheme for environmental ARGs monitoring. Declarations Funding sources: This research was funded by the Clinical Project of Changzhou Medical Center of Nanjing Medical University (CZKYCMCC202317); the 14th Five-Year Plan for Health Talents(No. 2022CZBj096); Changzhou Key Laboratory of Pathogen Biology (No. CM20223016); Jiangsu Province key research and development plan social development project(No. BE2023694); the Jiangsu Provincial Health Committee project (No. BH2023060). 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Clin Microbiol Rev 13:559–570. https://doi.org/10.1128/cmr.13.4.559-570.2000 Shuai LJ, Zhe HY, Ling HM et al Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus Xia C, Yan R, Liu C et al (2024) Epidemiological and genomic characteristics of global blaNDM-carrying Escherichia coli. Ann Clin Microbiol Antimicrob 23:58. https://doi.org/10.1186/s12941-024-00719-x Mmatli M, Mbelle NM, Osei Sekyere J (2022) Global epidemiology, genetic environment, risk factors and therapeutic prospects of mcr genes: A current and emerging update. Front Cell Infect Microbiol 12:941358. https://doi.org/10.3389/fcimb.2022.941358 ‘ A, Musialowski M, Wolacewicz M et al (2022) Development and validation of novel PCR primers for identification of plasmid-mediated colistin resistance ( mcr ) genes in various environmental settings. J Hazard Mater 425:127936. https://doi.org/10.1016/j.jhazmat.2021.127936 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Oct, 2025 Reviews received at journal 26 Oct, 2025 Reviews received at journal 14 Oct, 2025 Reviewers agreed at journal 28 Sep, 2025 Reviewers agreed at journal 25 Sep, 2025 Reviewers invited by journal 25 Sep, 2025 Editor assigned by journal 20 Sep, 2025 Submission checks completed at journal 19 Sep, 2025 First submitted to journal 19 Sep, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7655921","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":524920611,"identity":"7ab0c248-a8d0-4064-8b5c-b6b5737c3bdc","order_by":0,"name":"Yunqian Zhou","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yunqian","middleName":"","lastName":"Zhou","suffix":""},{"id":524920612,"identity":"729742ca-241d-4181-982f-470b1a0abc30","order_by":1,"name":"Fengming Wang","email":"","orcid":"","institution":"Changzhou Centre for Disease Control and 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15:05:23","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135614,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7655921/v1/4ae9c1d65286426138750a13.html"},{"id":93056662,"identity":"e1947533-006c-459c-983c-26224e936fe5","added_by":"auto","created_at":"2025-10-08 15:05:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":129583,"visible":true,"origin":"","legend":"\u003cp\u003eARGs detection mechanism using the LFD-RPA assay. Forward and reserse primers initiate RPA to amplify the target sequence. The FAM-labeled nfo probe is cleaved at the THF site by the nfo nuclease , resuliting a longer 5′ fragment and a shorter fragment with a 3′-end block. The 5′fragment serves as a new primer for extension, resulting in a double-labeled product containing both biotin and FAM. The amplified product is captured on the lateral flow strip by gold-conjugated anti-FAM antibodies and streptavidin, producing a positive test line.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7655921/v1/e61c4d2241e6dd3a89a99b65.png"},{"id":93056663,"identity":"a24750df-98fc-4467-b13f-668e2744e132","added_by":"auto","created_at":"2025-10-08 15:05:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":311409,"visible":true,"origin":"","legend":"\u003cp\u003eSensitivity of the LFD-RPA asssay for\u003cem\u003e bla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e\u003cem\u003e、mcr\u003c/em\u003e、\u003cem\u003etetX1\u003c/em\u003e、\u003cem\u003etetX2\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Results of lateral flow assay for ten-fold serial dilutions of the ten primer-probe sets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB \u003c/strong\u003eStandardize the grayscale values of the T-line and C-line in each group of test results to construct a calibration curve.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7655921/v1/9ae2a395cf7eeac8ea7e1a03.jpg"},{"id":93056666,"identity":"e9f802f2-76b5-4689-8cc4-5a3d95f44772","added_by":"auto","created_at":"2025-10-08 15:05:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":827868,"visible":true,"origin":"","legend":"\u003cp\u003eSpecificity analysis of the LFD-RPA asssay for \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e、\u003cem\u003emcr\u003c/em\u003e、\u003cem\u003etetX1\u003c/em\u003e、\u003cem\u003etetX2\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e genes\u003c/p\u003e\n\u003cp\u003e(T1: Klebsiella pneumoniae, T2: Pseudomonas aeruginosa, T3: Shigella, T4:Staphylococcus aureus, T5： Serratia marcescens, T6: Enterobacter cloacae, T7: corresponding gene positive plasmid control)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7655921/v1/af7e6f00559f9b82953209ae.png"},{"id":93057586,"identity":"3007108c-c74d-401f-b6a8-ea901e564a56","added_by":"auto","created_at":"2025-10-08 15:13:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":127081,"visible":true,"origin":"","legend":"\u003cp\u003eReproducibility test results of the LFD-RPA asssay for \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e、\u003cem\u003emcr\u003c/em\u003e、\u003cem\u003etetX1\u003c/em\u003e、\u003cem\u003etetX2\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e genes.Each group tests ten replicates.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7655921/v1/b9ed6c02fef2ca1e51771a1d.jpg"},{"id":93059506,"identity":"ec10a17f-cb47-46c0-abd0-ddb944b4dea7","added_by":"auto","created_at":"2025-10-08 15:37:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1956195,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7655921/v1/f13f05f5-53fa-4c63-af29-35fd1c05a673.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Visual detection for environmental antibiotic resistance genes by recombinase polymerase amplification combined with lateral flow dipstick","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe intensive dependence on antimicrobial agents has caused the prevalence of antimicrobial resistance (AMR) become increasingly critical, with microorganisms frequently harboring multiple antimicrobial resistance genes (ARGs)[1, 2]. Antibiotics are utilized not only in clinical therapeutics but also extensively in veterinary medicine and aquaculture, resulting in widespread environmental dissemination of residual compounds[3]. Low-level antibiotic exposure induces microbial resistance and facilitates the horizontal transfer of resistance genes via mobile genetic elements across species, thereby accelerating the dissemination of antimicrobial resistance[4, 5].In 2024, The World Health Organization has identified carbapenem-resistant Enterobacterales and Acinetobacter baumannii as the most problematic multidrug-resistant bacteria [6]. Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM) are common carbapenemases encoded by the \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e genes, respectively[7], and are capable of clonal transmission between humans and the environment[8].The increasing incidence of carbapenem-resistant bacteria and the paucity of novel antimicrobials have heightened dependence on colistin as a last-resort antibiotic, thereby driving the emergence of the colistin resistance[9].\u0026nbsp;And\u0026nbsp;the coexistence of carbapenemase genes and mobile colistin resistance (mcr) genes in the same bacterial isolates can lead to multidrug resistance(MDR)[10].Moreover, the tigecycline served as the final line of defense against severe infections by pan-drug-resistant bacterial pathogens has been challenged by the recent emergence of the mobile tet(X) orthologs that can confer tigecycline resistance\u0026nbsp;[11]. Therefore, carbapenem antibiotics exhibit widespread resistance, with recent emergence of colistin and tigecycline resistance, emphasizing the urgent need for rapid detection methods to improve antimicrobial resistance surveillance and clinical intervention.\u003c/p\u003e\n\u003cp\u003eConventional antimicrobial susceptibility testing (AST) methods, such as disk diffusion, agar dilution and broth microdilution are limited by phenotypic screening and prolonged detection cycles[12], whereas genotypic detection methods offer higher sensitivity and specificity, significantly reducing the time for resistance gene detection[13]. However, most genotyping detection methods depend on thermal cycling nucleic acid amplification requiring specialized laboratory equipment, limiting their applicability in point-of-care testing (POCT). To overcome this limitation, isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), and recombinase polymerase amplification (RPA) , have been developmented[14]. RPA, acknowledged as a viable alternative to polymerase chain reaction (PCR) for nucleic acid detection, is an innovative isothermal amplification technique that emulates the intracellular nucleic acid replication process, leveraging the replication mechanism of T4 bacteriophage[15].This technique relies on three key enzymes: a recombinase that binds single-stranded nucleic acids, single-strand DNA-binding protein (SSB), and strand-displacing DNA polymerase, all of which remain active at ambient temperature, with optimal reaction conditions between 37°C and 42°C. RPA amplification reactions have been demonstrated to achieve exponential growth of target genes in a very short time. RPA technology, when used with fluorescent probes, can monitor amplification in real time. It can also be integrated with lateral flow strips, simplifying workflows and reducing costs, making it suitable for POCT.\u003c/p\u003e\n\u003cp\u003eIn this study, a visualisation method for ambient temperature amplification targeting five antibiotic resistance genes (\u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e,\u003c/sub\u003e \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e,\u003c/sub\u003e \u003cem\u003emcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e, and \u003cem\u003etetX2\u003c/em\u003e) was established by combining RPA and lateral flow dipstick (LFD). This method provides a novel approach and methodology for the rapid detection of antibiotic resistance genes in environmental microorganisms.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e2.1 Bacterial Strain Collection and Identification\u003c/p\u003e\n\u003cp\u003eA total of 339 bacterial isolates were obtained from environmental samples collected within local hospitals in Changzhou from 2012 to 2021. The sampling, collection, and microbiological analysis procedures were conducted in accordance with the standards outlined in GB15982 \u0026quot;Hygienic standard for disinfection in hospitals\u0026quot; (2012).All strains were identifided utilizing a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) system (VITEK-MS, BioM\u0026eacute;rieux, France).\u003c/p\u003e\n\u003cp\u003e2.2 Phenotypic and genotypic antimicrobial resistance of strains\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We selected Klebsiella pneumoniae and Escherichia coli isolates for subsequent microbiological characterization.The reference method for\u0026nbsp;AST\u0026nbsp;was the microdilution method standardized by \u0026ldquo;Clinical and Laboratory Standards Institute\u0026rdquo;(CLSI)[16],utilizing an automated antimicrobial susceptibility platform(Thermo Fisher Scientific, USA)\u0026nbsp;. The antibiotics evaluated included ampicillin, piperacillin, cefuroxime, ceftriaxone, cefepime, levofloxacin, imipenem, meropenem, ertapenem, polymyxin, and tigecycline. Subsequently, the multi-drug-resistant strains of K. pneumoniae and E. coli, which demonstrated resistance phenotypes, were selected as the subjects for the study.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e, bla\u003csub\u003eKPC\u003c/sub\u003e, mcr, tetX1, and tetX2\u003c/em\u003e genes carried by multi-drug resistant K. pneumoniae and E. coli were detected by PCR, with amplicon visualization conducted through 1% agarose gel electrophoresis. The products were sent to Genewiz for Sanger sequencing, and the obtained gene sequences were validated for sequence fidelity through BLAST analysis against the GenBank database.\u003c/p\u003e\n\u003cp\u003e2.3 Primer and probe design\u003c/p\u003e\n\u003cp\u003eConserved regions from 22 \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e genotypes, including \u003cem\u003ebla\u003csub\u003eNDM-1\u003c/sub\u003e\u003c/em\u003e,\u003cem\u003ebla\u003csub\u003eNDM-2\u003c/sub\u003e\u003c/em\u003e,\u003cem\u003ebla\u003csub\u003eNDM-3\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003ebla\u003csub\u003eNDM-4\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003ebla\u003csub\u003eNDM-5\u003c/sub\u003e\u003c/em\u003e,and\u0026nbsp;\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-6\u003c/sub\u003e(GenBank accession numbers KX999121.1, KU510393.1,\u0026nbsp;JQ734687.1, KP772213.1, KP772211.1, NG049338.1, etc.), were selected through sequence alignment as the template for the RPA of the \u003cem\u003ebla\u003csub\u003eNDM\u0026nbsp;\u003c/sub\u003e\u003c/em\u003egene. Addtionally, conserved sequences from various genotypes of the \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e,\u003cem\u003e\u0026nbsp;mcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;tetX2\u003c/em\u003e genes were also employed as templates for RPA. The amplified target sequences were cloned into the pUC-18 plasmid vector to generate specific recombinant constructs. The PCR primer sequences used for plasmid construction are listed in Table 1.\u003c/p\u003e\n\u003cp\u003eIn this study, 10 sets of RPA primers were designed to target the \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e ,\u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e,\u003cem\u003e\u0026nbsp;mcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;tetX2\u0026nbsp;\u003c/em\u003egenes, along with nfo fluorescent probes for the LFD-RPA, designated as A1, A2, B1, B2, C1, C2, D1, D2, E1, and E2. The RPA primer pairs are optimized to a length of 30-35 bp, with the 5\u0026rsquo; end of the reverse primers labeled with biotin. The nfo probes are 48-55 bp, and a tetrahydrofuran (THF) group replaces the adenylate site\u0026nbsp;as the recognition site of endonuclease, located at least 30 bp away from the 5\u0026prime; end and 15 bp away from the 3\u0026rsquo; end. Moreover,the 5\u0026apos; end of the probe is labeled with a FAM fluorophore, while the 3\u0026rsquo;end is modified with a C3 spacer to prevent extension. Both primers and probes were synthesized by\u0026nbsp;Genewiz, as detailed in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e PCR Primer Table for \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e\u003csub\u003e、\u003c/sub\u003e\u003cem\u003eblaKPC\u003c/em\u003e, \u003cem\u003emcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e and \u003cem\u003etetX2\u003c/em\u003e genes\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eSequence (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.066%;\"\u003e\n \u003cp\u003eproducts(bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003e\u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eCATTAGCCGCTGCATTGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 21.066%;\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003e\u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eGCAAGCTGGTTCGACAACGC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003e\u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eCATTCAAGGGCTTTCTTGCTGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 21.066%;\"\u003e\n \u003cp\u003e538\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003e\u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eACGACGGCATAGTCATTTGC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003e\u003cem\u003emcr\u003c/em\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eCGGTCAGTCCGTTTGTTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 21.066%;\"\u003e\n \u003cp\u003e309\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003e\u003cem\u003emcr\u003c/em\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eCTTGGTCGGTCTGTAGGG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003e\u003cem\u003etetX1\u003c/em\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eCGAAAAATGTTGCTTGGCAGCTT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 21.066%;\"\u003e\n \u003cp\u003e486\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003e\u003cem\u003etetX1\u003c/em\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eAGTTGTTGAACGAATTAACTCC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003e\u003cem\u003etetX2\u003c/em\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eCGGGATGTCCAAGGTAAGAAAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 21.066%;\"\u003e\n \u003cp\u003e343\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.736%;\"\u003e\n \u003cp\u003e\u003cem\u003etetX2\u003c/em\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.198%;\"\u003e\n \u003cp\u003eTGACAACGTCGTATGAATCAA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e The Primer and Probe Sequences for LFD-RPA\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"612\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePrimer direction\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSequence(5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026nbsp;\u003c/sub\u003eA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCTTATGCCAATGCGTTGTCGAACCAGCTTGCCC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBiotin-CCCAACGGTGATATTGTCACTGGTGTGGCC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eprobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5`6-FAM-CAACACAGCCTGACTTTCGCCGCCAATGGCTG/idSp/GTCGAACCAGCAACCGCG-3`C3-Spacer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026nbsp;\u003c/sub\u003eA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCATTAGCCGCTGCATTGATGCTGAGCGGGTGCATGCCC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBiotin-CCCTGACGATCAAACCGTTGGAAGCGACTGCCC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eprobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5`6-FAM-AGCTCGCACCGAATGTCTGGCAGCACACTTCC/idSp/ATCTCGACATGCCG-3`C3-Spacer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026nbsp;\u003c/sub\u003eB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTGCCACCGCGCTGACCAACCTCGTCGCGGAAC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBiotin-AAGCCCTTGAATGAGCTGCACAGTGGGAAGCG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eprobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5`6-FAM-GACTTTGGCGGCTCCATCGGTGTGTACGCGATGGA/idSp/ACCGGCTCAGGCGCAACTGTAAGTTA-3`C3-Spacer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026nbsp;\u003c/sub\u003eB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAGGAGCGCTTCCCACTGTGCAGCTCATTCAAGGGCT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBiotin-TCATGCCTGTTGTCAGATATTTTTCCGAGATG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eprobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5`6-FAM-TGTGCTGGCTCGCAGCCAGCAGCAGGCCGGCTTGC/idSp/GGACACACCCATCCGTTACGGCA-3`C3-Spacer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003emcr\u0026nbsp;\u003c/em\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCTAAAGCCTGTGTTGATTTTGCTATTAATCATGGG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBiotin-CCCAATCGGCGCATCAAACCCTTGCCCCAA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eprobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5`6-FAM-CGGTCTATGATACGACCATGCTCCAAAATGC/idSp/CTACAGACCGACCAAGCCGAG-3`C3-Spacer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003emcr\u0026nbsp;\u003c/em\u003eC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAGACGCGGTACAAGCAACCAAGCCTGATATGCG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBiotin-TGGTCACGCCATCGATCTTGGCAAGCTGTGG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eprobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5`6-FAM-CGTCGTCGGTGAGACGGCACGCGCCGATCATG/idSp/TCAGCTTCAATGGCTATGA-3`C3-Spacer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003etetX1\u0026nbsp;\u003c/em\u003eD1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTTAAAAGGTTACTCCTATTTGGGATTATAATA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBiotin-TATGGAGCCAATAAGACCAAACGCTTTACC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eprobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5`6-FAM-CTGGCTCGATTTATTCAAGGAGCTGGTGCAGC/idSp/GCATTTCCAGCACTCGTG-3`C3-Spacer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003etetX1\u0026nbsp;\u003c/em\u003eD2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTTATTGGCTCCATAGTAGCTATGGGAGAAG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBiotin-AGTATAATTCCTTTGATATCAAAATGACCTT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eprobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5`6-FAM-TTCTACTCATTCCTATGATAACAATTATCACTGT/idSp/CCGTTTCTTATGAAATTATTAA-3`C3-Spacer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003etetX2\u0026nbsp;\u003c/em\u003eE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGCTTGCACTTTATGCGTTAATGCAGGTTATCT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBiotin-CAAACGGCCTAAATACAGCATCCAAAGCGCACT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eprobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5`6-FAM-TCTGACCGATTTGGTCGGCGCCCAGTGCTGTTG/idSp/TGTCATTAATAGGCGCATCGCTG-3`C3-Spacer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003etetX2\u0026nbsp;\u003c/em\u003eE2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCATGAGCAAGGTGCTTTACAGGGATTATTG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBiotin-CTAAACCAATAATCCAAATCCAGCCATCCCA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eprobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5`6-FAM-GCAACCGGTGTTATTGGCCCATTACTGTTTAC/idSp/GTTATTTATAATCATTCAC-3`C3-Spacer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e2.4 Establishment of the LFD-RPA reaction system\u003c/p\u003e\n\u003cp\u003eAmplification assays targeting plasmid containing bla\u003cem\u003e\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e,\u003cem\u003e\u0026nbsp;mcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;tetX2\u003c/em\u003e genes were conducted using the primer pairs and probes detailed in Table 2. The RPA reactions were carried out using the TwistAmp nfo kit (TwistDX, UK). The reaction mixture consisted 2.1\u0026nbsp;\u0026mu;l of the upstream primer (10\u0026nbsp;\u0026mu;M), 2.1 \u0026mu;l of the\u0026nbsp;downstream primer (10 \u0026mu;M), 0.6 \u0026mu;l of the nfo probe (10 \u0026mu;M), 29.5 \u0026mu;l of Rehydration Buffer, and a total of 13.2 \u0026mu;l of DNA template and nuclease-free water, yielding a final volume of 47.5 \u0026mu;l. Immediately, 2.5 \u0026mu;l of 280 mM magnesium acetate was added into the system, and the reaction was incubated at 37\u0026ndash;39\u0026deg;C for 20\u0026ndash;40 minutes. Subsequently, the RPA reactions were performed on a real-time PCR system\u0026nbsp;(QuantStudio 5, ABI)\u0026nbsp;with an isothermal cycling protocol: an initial pre-denaturation at 40\u0026deg;C for 1 minute, followed by 40 cycles of amplification at 40\u0026deg;C for 31 seconds. Then, 5\u0026nbsp;\u0026mu;L of the amplification product was diluted into 95 \u0026mu;L of PBST to prepare the lateral flow immunoassay. The prepared mixture was applied to a lateral flow strip embedded with anti-FAM antibodies, using the HybriDetect - Universal Lateral Flow Assay Kit(Milenia Biotec,Germany), with results interpreted within 5 minutes.\u003c/p\u003e\n\u003cp\u003e2.5 Sensitivity of the LFD-RPA assay\u003c/p\u003e\n\u003cp\u003eThe recombinant plasmisds were converted to copy numbers by measuring the concentration of plasmids , following the formula: DNA copies/\u0026mu;L = (ng/\u0026mu;L \u0026times; 6.02 \u0026times; 10\u003csup\u003e23\u0026nbsp;\u003c/sup\u003e\u0026times; 10\u003csup\u003e\u0026minus;9\u0026nbsp;\u003c/sup\u003e)/(Fragment length (bp)\u0026times;660).Starting with a DNA template concentration of 40 ng/mL, the plasmids were serially diluted ten-fold. The prepared \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e( 1.764\u0026times;10\u003csup\u003e9\u0026nbsp;\u003c/sup\u003ecopies/mL-1.764\u0026times;10\u003csup\u003e0\u0026nbsp;\u003c/sup\u003ecopies/mL),\u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e(1.800\u0026times;10\u003csup\u003e9\u0026nbsp;\u003c/sup\u003ecopies/mL-1.800\u0026times;10\u003csup\u003e0\u003c/sup\u003e copies/mL),\u003cem\u003e\u0026nbsp;mcr\u003c/em\u003e(1.803\u0026times;10\u003csup\u003e9\u003c/sup\u003e copies/mL-1.803\u0026times;10\u003csup\u003e0\u0026nbsp;\u003c/sup\u003ecopies/mL),\u003cem\u003e\u0026nbsp;tetX1\u003c/em\u003e(1.061\u0026times;10\u003csup\u003e9\u003c/sup\u003e copies/mL-1.061\u0026times;10\u003csup\u003e0\u003c/sup\u003e copies/mL), and \u003cem\u003etetX2\u003c/em\u003e(1.271\u0026times;10\u003csup\u003e9\u003c/sup\u003e copies/mL-1.271\u0026times;10\u003csup\u003e0\u003c/sup\u003e copies/mL)\u0026nbsp;genes\u0026nbsp;were utilized for sensitivity testing, and different copy numbers of the genes were used as templates for LFD-RPA.The limit of detection (LOD) of the five genes were determined via observation of the color intensity of the dipsticks test line.\u003c/p\u003e\n\u003cp\u003e2.6 Specificity of the LFD-RPA Assay\u003c/p\u003e\n\u003cp\u003ePhenotypic isolates exhibiting resistance to non-KPC, NDM, MCR, and TETX1/2 among K. pneumoniae, Pseudomonas aeruginosa, Shigella spp., Staphylococcus aureus, Sphingomonas paucimobilis, and Enterobacter cloacae were obtained through routine epidemiological surveillance conducted by Changzhou Disease Control and Prevention Centre. The strains were performed using LFD-RPA reactions separately, and the results were compared with those of the positive plasmid control groups to evaluate the specificity of the LFD-RPA system.\u003c/p\u003e\n\u003cp\u003e2.7 Reproducibility of the LFD-RPA assay\u003c/p\u003e\n\u003cp\u003eWe Selected the amplification systems with higher sensitivity from Table 2 and perform ten consecutive repetitions of LFD-RPA. The reproducibility of the LFD-RPA amplification is evaluated by analyzing the positive rate across these replicates.\u003c/p\u003e\n\u003cp\u003e2.8 Statistical analysis\u003c/p\u003e\n\u003cp\u003eGrayscale image analysis of the test line on the lateral flow strip was conducted using ImageJ software to quantify the grayscale value. The results were expressed as the mean \u0026plusmn; standard deviation (SD), and all statistical analyses were performed using the SPSS 26.0 package.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Identification of bacterial strains and antimicrobial resistance genes\u003c/p\u003e\n\u003cp\u003eThrough identification using VITEK-MS and AST via microdilution, a total of 65 isolates of K. pneumoniae and E. coli strains carrying genes for carbapenems, tigecycline, and polymyxins were detected. Among these, three isolates harbored the \u003cem\u003ebla\u003csub\u003eNDM\u0026nbsp;\u003c/sub\u003e\u003c/em\u003egene, eleven carried the\u003cem\u003e\u0026nbsp;bla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e gene, one possessed the \u003cem\u003emcr\u003c/em\u003e gene, and one contained both the \u003cem\u003etetX1 and tetX2\u003c/em\u003e genes.\u003c/p\u003e\n\u003cp\u003e3.2 Schematic of the LFD-RPA detection process\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe schematic workflow for the detection of ARGs is depicted in Figure 1. The extracted genomic DNA functions as the template for RPA amplification, with the nfo probe conjugated with FAM at the 5\u0026apos; end and the reverse primer labeled with biotin. Consequently, a positive RPA reaction yields dual-labeled amplicons that specifically bind to streptavidin immobilized on the Test line (T line), producing a visible signal. Simultaneously, the Control line (C line) also shows a colorimetric response. In the case of a negative result, the FAM-labeled probe is captured by anti-FAM antibodies, while the resulting nucleic acid fragments, lacking biotin, cannot bind to streptavidin, resulting in no visible band at the T line.\u003c/p\u003e\n\u003cp\u003e3.3 Sensitivity analysis of the LFD-RPA assay\u003c/p\u003e\n\u003cp\u003eThe sensitivity of the LFD-RPA was analyzed using various copies of recombinant plasmids containing the \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003emcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e, and \u003cem\u003etetX2\u003c/em\u003e genes, all with an initial concentration of 40 ng/mL. The highest number of copies at which a positive result was observed was designated as the LOD. Lateral flow assay results are shown in Figure 2, with the \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e gene exhibiting a maximum detectable dilution of 10\u003csup\u003e8\u003c/sup\u003e, corresponding to the LOD of 1.764\u0026nbsp;\u0026times;10\u003csup\u003e1\u0026nbsp;\u003c/sup\u003ecopies/mL. Results for other genes are detailed in Table 3 and Figure 2. Except for the \u003cem\u003emcr\u003c/em\u003e gene, which had a higher detection limit of 1.803\u0026times;10\u003csup\u003e2\u003c/sup\u003ecopies/mL, the LOD of the nfo-RPA amplification for the other genes ranged between 10 and 20 copies/mL, with \u003cem\u003etetX1\u0026nbsp;\u003c/em\u003edemonstrating the highest sensitivity at 1.061\u0026times;10\u003csup\u003e1\u003c/sup\u003e copies/mL. Figure 2B also showed that among the ten sets of primers and nfo-probes, the specific primer and probe sets that were categorized within groups A1, B1, C2, D2, and E2 demonstrated a significantly higher level of sensitivity, with grayscale value exhibiting a linear correlation with template concentration. Notably, the A1 set targeting \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e exhibited the minimal slope in the graysclae value versus dilution curve, indicating sustained high amplification efficiency at low template concentrations. These results further validated the ultra-high sensitivity and practical applicability of nfo-RPA for rapid on-site detection of resistance genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Detection limits of LFD-RPA in different amplification systems\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"465\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003egene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003eLOD（copise/ mL）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003emaximum detectable dilution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eamplification systems\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.764\u0026times;10\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eA2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.800\u0026times;10\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eB1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003emcr\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;1.803\u0026times;10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003etetX1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.061\u0026times;10\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eD2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cem\u003etetX2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e1.271\u0026times;10\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 154px;\"\u003e\n \u003cp\u003e10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eE2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e3.4 Specificity and reproducibility analysis of LFD-RPA assay\u003c/p\u003e\n\u003cp\u003eStrains collected in the routine antimicrobial resistance surveillance work were subjected to the Specificity analysis for the LFD-RPA assays. The isolates included K. pneumoniae strains exhibiting resistance phenotypes absent of \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003emcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e, or \u003cem\u003etetX2\u003c/em\u003e genes (resistance profile: AMP-ETP-CTX-CAZ-CIP positive, designated as T1), P.aeruginosa susceptible strains (T2), Shigella (SXT-AZM-E-CIP-AZT, T3), S. aureus (OXC-ERY-LEV-TET-GEN-VAN, T4), S. \u0026nbsp;marcescens (C-CAZ, T5), and E. cloacae (CAZ-AZT-SAM-CZO-TZP, FEP, T6). These isolates, along with corresponding plasmid controls containing the target resistance genes (T7), went through the specificity validation across five distinct LFD-RPA platforms. The results presented in Figure 3 illustrated that the LFD-RPA assays targeting the \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003emcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e, and \u003cem\u003etetX2\u003c/em\u003e genes demonstrated high specificity for their respective resistance phenotypes, with no false-positive amplifications observed.\u003c/p\u003e\n\u003cp\u003eTo evaluate the reproducibility of these assays, the most sensitive primer and probe combinations from ten selected assays were tested using ten-fold serial dilutions of the respective plasmid templates, with each assay performed in ten replicates. The findings, depicted in Figure 4, showed consistent positive detection across all replicates, confirming the robustness and reliability of the LFD-RPA methodology for resistance gene detection.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCurrently, the rapid dissemination of antimicrobial-resistant microorganisms has emerged as one of the foremost global threats to human health. ARGs have transcended clinical boundaries, spreading extensively across aquatic systems, soils, the atmosphere, and the food chain[17]. Consequently, ARG-mediated environmental contamination has become a significant public health concern worldwide.Mobile genetic elements (MGEs) such as plasmids, integrons and transposons facilitate the horizontal transfer of critical resistance genes such as \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e, thereby promoting the dissemination of ARGs within bacterial communities and enhancing the spread of AMR[18].The proliferation of ARGs in environmental matrices affects ecological integrity and presents potential public health hazards via contaminated potable water, food sources, and aerosols[19].Therefore, the implementation of rapid, on-site monitoring of environmental antimicrobial resistance genes is of critical importance for the prevention and control of the dissemination of antibiotic resistance.This study focuses on five representative ARGs: \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003emcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e, and \u003cem\u003etetX2\u003c/em\u003e develop a visual detection method based on LFD-RPA for ambient temperature application.Comprehensive validation encompassing sensitivity, specificity, and reproducibility analyses confirms that this methodology offers a practical and readily deployable platform for environmental surveillance of antimicrobial resistance.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBla\u003csub\u003eNDM\u0026nbsp;\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003ebla\u003csub\u003eKPC\u0026nbsp;\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003emcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e, and \u003cem\u003etetX2\u003c/em\u003e represent the key resistance determinants for the three major last-resort antibiotic classes: carbapenems, polymyxins, and tigecycline. These genes play a critical role in clinical and environmental antimicrobial resistance studies, and their presence and spread pose a substantial threat to public health[20].For instance, the metallo-β-lactamases and serine carbapenemases encoded by the \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e and \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e genes can hydrolyze virtually all\u0026nbsp;β-lactam antibiotics, resulting in bacterial pan-resistance[21, 22],The mcr gene family confers colistin resistance through the modification of lipid A , with colistin serving as the ultimate therapeutic choice for treating carbapenem-resistant Gram-negative bacterial infections[23].Enzymes encoded by \u003cem\u003etetX1\u003c/em\u003e and \u003cem\u003etetX2\u003c/em\u003e belong to the TetX enzyme family, which catalyzes the hydroxylation of tetracycline-class antibiotics (including tigecycline), resulting in their conversion to inactive metabolites and thereby mediating bacterial tigecycline resistance[24].The coexistence of these five genes within the same environmental micro-ecological niche may induce multidrug resistance and potentially disseminate to pathogenic bacteria via horizontal gene transfer, resulting in a clinical scenario where therapeutic options become ineffective[25].Therefore, implementing joint surveillance of these genes is crucial for assessing AMR risks in the environment and formulating subsequent targeted intervention strategies.\u003c/p\u003e\n\u003cp\u003ePCR, multiplex PCR, and real-time quantitative PCR have been widely employed in the detection of ARGs[26, 27]. However, these conventional approaches rely on gel electrophoresis for result interpretation or necessitate specialized real-time fluorescence quantification instruments, which are not only time-consuming but also demand advanced laboratory equipment[28].To address this challenge, this study established a LFD-RPA system utilizing the nfo probe, to fulfill POCT requirements. RPA enables exponential amplification at 37–42°C without thermal cycling. By incorporating dual labeling with FAM-biotin into the amplification products via the nfo probe, RPA products are captured on a lateral flow strip by anti-FAM gold-labeled antibodies, forming a visually detectable Test line, which can be interpreted within 5 minutes[29]. Compared to traditional PCR, the LFD-RPA method eliminates the need for costly equipment, complex optical systems, and data analysis software,with the entire process can be performed at room temperature, offering a simple operation and shorter turnaround time\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Designing primers and probes is the most critical and challenging step in establishing RPA amplification. In this study, we meticulously designed ten primer-probe sets targeting five representative ARGs. Specifically, for the \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u0026nbsp;\u003c/em\u003egene, we considered 22 different variants, including \u003cem\u003ebla\u003csub\u003eNDM-1\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003ebla\u003csub\u003eNDM-5\u003c/sub\u003e\u003c/em\u003e, and \u003cem\u003ebla\u003csub\u003eNDM-9\u003c/sub\u003e\u003c/em\u003e. A previous research has indicated that these three variants are the most prevalent among \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u0026nbsp;\u003c/em\u003efamilyand are widely distributed globally, particularly across Asia, Europe, and North America[30]. Consequently, the primers and probes designed for the \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u0026nbsp;\u003c/em\u003egene in this study exhibit both broad representativeness and applicability.The \u003cem\u003emcr-1\u0026nbsp;\u003c/em\u003egene is globally recognized as the most widely reported plasmid-mediated colistin resistance gene ,and the \u003cem\u003emcr-3\u003c/em\u003e gene has been frequently detected in Thailand, Vietnam, and China, particularly in Escherichia coli isolates from clinical and agricultural sources[31]. This study involved the development of two sets of primers and probes which precisely target conserved sequence from \u003cem\u003emcr-1\u003c/em\u003e to\u003cem\u003e\u0026nbsp;mcr-10\u003c/em\u003e, thereby ensuring comprehensive and accurate detection. The success of RPA amplification critically depends on the selection of conserved regions within the respective ARGs, as well as the sensitivity, reproducibility, and specificity of the primers and probes. Consequently, the primers and probes were subjected to rigorous experimental validation and optimization, as detailed below.\u003c/p\u003e\n\u003cp\u003eFigure 2 illustrates the visualization of plasmid templates in LFD-RPA at 10-fold gradient dilutions. As the template concentration decreases, the color of the test line transitions gradually from deep red to a lighter shade. Nevertheless, it remains discernible by the naked eye even at the furthest right, highlighting the exceptional sensitivity of the system. Through grayscale scanning and subsequent conversion of copy numbers, LOD were determined as follows: \u003cem\u003etetX1\u003c/em\u003e at 1.061×10\u003csup\u003e1\u003c/sup\u003ecopies/mL, \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e at 1.764×10\u003csup\u003e1\u003c/sup\u003e copies/mL, \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e at 1.800×10\u003csup\u003e1\u003c/sup\u003e copies/mL, and \u003cem\u003etetX2\u003c/em\u003e at 1.271×10\u003csup\u003e1\u003c/sup\u003e copies/mL. The LOD for \u003cem\u003emcr\u0026nbsp;\u003c/em\u003ewas slightly higher at 1.803×10\u003csup\u003e2\u003c/sup\u003e copies/mL, yet notably lower compared to the study of Gorecki et al. In their study, researchers utilized quantitative PCR to monitor \u003cem\u003emcr\u003c/em\u003e genes in the environment, detecting \u003cem\u003emcr-1\u003c/em\u003e at 9.35×10\u003csup\u003e2\u003c/sup\u003e copies/mL and \u003cem\u003emcr-3\u003c/em\u003e at 3.17×10\u003csup\u003e4\u003c/sup\u003e copies/mL[32].In the specificity experiment(Figure 3), six clinical isolates (T1-T6) lacking the target gene but possessing other drug resistance profiles, along with five positive plasmids (T7) containing the detection gene, were selected. Results revealed the absence of T-lines for T1-T6, with only colored C-lines observed. In contrast, T-lines for T7 were clearly visible, demonstrating the system's 100% specificity without cross-reaction to non-target genes. Fig. 4 showed that reproducibility tests\u0026nbsp;were repeated 10 times with the same batch and the same operator, using 10\u003csup\u003e7\u003c/sup\u003e-fold diluted plasmid as template. The 10 T lines had the same depth, ImageJ gray scanning showed CV \u0026lt;5%, indicating that the system had excellent intra-batch repeatability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe remarkable characteristic of this study is that the established amplification method for detecting environmental resistance genes can be advanced and visible at room temperature without relying on complex large-scale instruments and equipment, thus effectively realizing the portability and miniaturization of detection equipment. This portable and compact design greatly meets the need for rapid response in environmental sample detection. Through this technical means, we have not only significantly improved the speed of testing, but also significantly reduced the economic cost of testing, making rapid on-site detection of antibiotic resistance genes feasible.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eTo conclude , this study successfully designed primers and probes sets targeting five key ARGs including \u003cem\u003ebla\u003csub\u003eNDM\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003ebla\u003csub\u003eKPC\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003emcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e and \u003cem\u003etetX2\u003c/em\u003e , and developed a LFD-RPA system achieved visual detection at room temperature with sensitivity of 1.061×10\u003csup\u003e1\u003c/sup\u003e-1.803×10\u003csup\u003e2\u003c/sup\u003ecopies/mL, specificity of 100% and reliable repeatability. It breaked through the dependence on instruments and laboratories, combined the requirements of high sensitivity and field ease of use, and provided a feasible new scheme for environmental ARGs monitoring.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding sources:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Clinical Project of Changzhou Medical Center of Nanjing Medical University (CZKYCMCC202317); the 14th Five-Year Plan for Health Talents(No. 2022CZBj096); Changzhou Key Laboratory of Pathogen Biology (No. CM20223016); Jiangsu Province key research and development plan social development project(No. BE2023694); the Jiangsu Provincial Health Committee project (No. BH2023060). Jiangsu Province Schistosomiasis and Parasitic and Endemic Disease Prevention and Research Project(x202339).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePazda M, Kumirska J, Stepnowski P, Mulkiewicz E (2019) Antibiotic resistance genes identified in wastewater treatment plant systems \u0026ndash; A review. 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J Hazard Mater 425:127936. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jhazmat.2021.127936\u003c/span\u003e\u003cspan address=\"10.1016/j.jhazmat.2021.127936\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Antibiotic resistance gene, Recombinase polymerase amplification, Lateral flow dipstick, point-of-care testing","lastPublishedDoi":"10.21203/rs.3.rs-7655921/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7655921/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe emergence of the antibiotic resistance genes(ARGs) has posed a significant challenge in controlling the spread of multidrug-resistant bacteria both in clinic and environment.To establish the purpose of detecting the environmental ARGs in room temperature and achieving visualization, this study developed a rapid detection by combining recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) targeting crucial ARGs including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eNDM\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003e\u003cem\u003eKPC\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003emcr\u003c/em\u003e, \u003cem\u003etetX1\u003c/em\u003e and \u003cem\u003etetX2.\u003c/em\u003e Various primers and nfo probes were designed and LFD-RPA method was established, followed by the sensitivity, specificity and repeatability tests.Sensitivity was evaluated with ten-fold serial dilutions of plasmid standards, specificity was tested against environmental isolates lacking target genes, and reproducibility was assessed in ten replicates. Results showed that the limits of detection ranged from 1.061\u0026times;10\u003csup\u003e1\u003c/sup\u003e to 1.803\u0026times;10\u003csup\u003e2\u003c/sup\u003ecopies/mL,with \u003cem\u003etetX1\u003c/em\u003e demonstrating the highest sensitivity at 1.061\u0026times;10\u003csup\u003e1\u003c/sup\u003e copies/mL .The LFD-RPA system showed 100% specificity without cross-reactivity, and exhibited great repeatability. Thus, the LFD-RPA platform provided a highly sensitive, specific and one-site detection for real-time environmental monitoring of key ARGs.\u003c/p\u003e","manuscriptTitle":"Visual detection for environmental antibiotic resistance genes by recombinase polymerase amplification combined with lateral flow dipstick","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 15:05:18","doi":"10.21203/rs.3.rs-7655921/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-27T04:41:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T00:32:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-14T19:43:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299684581270515946811587555654363854982","date":"2025-09-28T18:09:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64495009910167963623070917797450095005","date":"2025-09-25T17:21:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-25T14:19:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-20T14:32:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-19T14:37:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Microbiology","date":"2025-09-19T07:46:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8b9eb2cf-58de-4c20-a796-8629deec9579","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-28T15:38:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 15:05:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7655921","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7655921","identity":"rs-7655921","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}