https://orcid.org/0000-0002-6061-1781
Figures
Abstract
Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen of significant clinical and public health concern, necessitating the development of rapid and reliable detection methods. Traditional diagnostic approaches, which rely on culture-dependent techniques and biochemical identification, are often labor-intensive, time-consuming, and technically demanding. This study describes a novel single-tube, two-step, rapid detection platform that integrates recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats-associated protein Cas12b technology. Through systematic experimental optimization, the study identified an optimal RPA primer pair (F2-R1) and single-guide ribonucleic acid 553 that targets the lasR gene of P. aeruginosa, with reaction conditions optimized at 42°C and a primer concentration of 10 μM. The RPA-clustered regularly interspaced short palindromic repeats/Cas12b fluorescence detection system (RPA-Cas12b-Fluo) demonstrated a sensitivity threshold of 10 copies of deoxyribonucleic acid (DNA) per reaction and a bacterial detection limit of 50 colony-forming units (CFU) per reaction. When coupled with a lateral flow strip (RPA-Cas12b-LFS), the sensitivity was slightly reduced but remained robust, achieving detection limits of 10² copies and 200 CFU per reaction. Specificity assays confirmed a high discriminatory capacity for P. aeruginosa with no cross-reactivity observed against P. fluorescens, P. putida, or six common foodborne pathogens, thereby validating the specificity profile of the platform. The applicability of the method was further validated by analyzing 20 water samples, which demonstrated 100% concordance with the national standard culture method. These findings have significant implications for improving outbreak surveillance and mitigating the risk of foodborne transmission associated with P. aeruginosa.
Citation: Lin H, Wang S, Xie Y, Cheng C, Jin J, Song X, et al. (2026) Single-tube two-step RPA-CRISPR/Cas12b platform for detection of Pseudomonas aeruginosa. PLoS One 21(3): e0340856. https://doi.org/10.1371/journal.pone.0340856
Editor: Yong Qi, Huadong Research Institute for Medicine and Biotechniques, CHINA
Received: April 12, 2025; Accepted: December 29, 2025; Published: March 18, 2026
Copyright: © 2026 Lin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This research was funded by Classification Development of Beijing Municipal Universities-Construction of Urban Agriculture and Forestry Characteristic Teacher Team, grant number 11000024T000002961733. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Pseudomonas aeruginosa, a ubiquitous environmental bacterium, is an opportunistic pathogen associated with cutaneous infections, particularly in individuals with burns, scalds, or ocular diseases, potentially leading to sepsis [1]. Chinese national standards GB 8537−2018 and GB 8538−2022 mandate the absence of P. aeruginosa in drinking water and specify its detection via membrane filtration, selective culture media, and biochemical tests. However, these traditional methods are laborious, time-consuming, and technically demanding [2]. There is a pressing need for detection technologies that are faster, more accurate, and less costly and technically demanding [3].
Isothermal nucleic acid amplification techniques have emerged as promising tools for the detection of microorganisms in food matrices, exhibiting distinct advantages such as simplicity, rapidity, high efficiency, high sensitivity, broad applicability, and minimal dependency on specialized equipment [4]. Prominent examples of these methods include rolling circle amplification [5], loop-mediated isothermal amplification (LAMP) [6], cross-priming amplification [7], and recombinase polymerase amplification (RPA) [3]. Among these, RPA has gained widespread application in molecular diagnostics, food safety testing, and environmental monitoring due to its operational simplicity and rapidity. Developed by Piepenburg et al. in 2006 and commercialized by TwistDx, RPA enables nucleic acid amplification at a constant temperature of 37–42°C, yielding target products within 30 min [8]. This technique requires minimal instrumentation, unlike other isothermal amplification methods, making it particularly suitable for rapid and low-resource diagnostic applications. Recent studies have expanded the utility of RPA for the detection of P. aeruginosa. Jin et al. [9] established an RPA-based method for the rapid detection of P. aeruginosa, demonstrating significantly shorter detection times than polymerase chain reaction (PCR) and real-time quantitative PCR, although with reduced sensitivity. Tran et al. [10] developed a multiplex RPA method that achieved high sensitivity without requiring genomic DNA extraction, thereby simplifying the workflow. Xu et al. [11] integrated RPA with magnetic nanobead-based DNA extraction to detect P. aeruginosa in clinical samples, such as blood, wound swabs, and sputum, achieving high sensitivity and specificity. Additionally, Yang et al. [12] combined the RPA with LFS to enable visual detection and further enhance its applicability in point-of-care settings. These advancements highlight the potential of RPA as a versatile platform for rapid and accurate microbial detection in diverse applications.
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system, first identified in Escherichia coli in 1987, has transformed molecular diagnostics since its adaptation as a programmable gene editing tool in 2012 [13]. Current diagnostic platforms primarily use Cas12a (Type V), Cas12b (Type V-B), and Cas13a (Type VI) nucleases, each exhibiting distinct molecular recognition mechanisms [14]. Cas12a demonstrates dual DNA/RNA targeting capacity and mature micro RNA discrimination but requires stringent CRISPR RNA-target complementarity (≥18 nt), shows reduced mismatch tolerance in PAM-distal regions, and is susceptible to nucleic acid secondary structures [14, 15]. In contrast, Cas12b achieves enhanced specificity through minimal single-guide RNA (sgRNA)-target mismatch tolerance (<3 nt) [16], coupled with broad thermotolerance (37–60°C) that enables seamless integration with RPA workflows [17,18]. Its trans-cleavage activity generates multiple DNA fragments per activation event, improving the detection sensitivity [19]. Cas13a leverages collateral RNA cleavage for signal amplification, enabling attomolar-level detection and single-nucleotide polymorphism discrimination [3]. However, nonspecific ribonuclease activity elevates background noise at low target concentrations, whereas CRISPR RNA-target mismatches critically impair cleavage efficiency [13]. These orthogonal CRISPR systems establish multiplexed diagnostic frameworks through PCR/RPA-mediated template amplification coupled with Cas-mediated sequence recognition [20], achieving <10 copies of the detection limit across clinical and environmental matrices [14,19]. The strategic selection of Cas effectors based on the target nucleic acid type, required specificity, and operational constraints enables an optimized assay design for diverse diagnostic applications. Compared with Cas13a, which only cleaves target RNA, and Cas12b, which relies on PCR for template amplification, the RPA-CRISPR/Cas12b detection method amplifies targets via RPA and achieves specific recognition through the Cas12b-sgRNA complex. This method simultaneously activates the trans-cleavage activity of Cas12b to cleave both target double-stranded DNA and nonspecific single-stranded DNA (ssDNA) [21]. It is suitable for quantitative nucleic acid detection in laboratories equipped with specialized instruments and can be adapted for on-site and primary laboratory settings using portable real-time fluorescence detectors or nucleic acid test strips for large-scale sample screening and detection [22].
Integrating the CRISPR-Cas system with RPA has emerged as a promising approach for detecting P. aeruginosa. Previous studies explored the combination of RPA with Cas12a and Cas13a. Zhang et al. [23] developed an RPA-CRISPR/Cas12a method targeting the lasB gene, using LFS to visualize the results. Yang et al. [24] established a one-tube RPA-CRISPR/Cas12a method for the lasB gene, while Liu et al. [25] have demonstrated improved sensitivity using fluorescence detection over agarose gel electrophoresis in an RPA-CRISPR/Cas12a method targeting the oprI gene. Zhu et al. [26] have reported a two-step RPA-CRISPR/Cas13a method for the mexX gene that showed enhanced sensitivity compared with single-step approaches. Previous studies targeting P. aeruginosa have predominantly utilized lasB, oprL, and mexX gene fragments. In contrast, the lasR gene-encoding the transcriptional regulator LasR, a central modulator of the P. aeruginosa quorum-sensing (QS) system governing virulence and biofilm formation [1], remains unexplored for diagnostic applications. Given its functional significance, evolutionary conservation across P. aeruginosa strains, and species-specific sequence features, lasR represents a biologically compelling target for detection.
In this study, we developed a novel single-tube, two-step detection platform for P. aeruginosa by integrating RPA with the CRISPR/Cas12b technology. The platform was designed to achieve rapid, sensitive, and specific detection of P. aeruginosa while minimizing technical complexity and contamination risks. The study objectives included the systematic optimization of primer pairs, sgRNA sequences, and reaction conditions to enhance analytical sensitivity and specificity. Additionally, the specificity of the platform was validated against closely related Pseudomonas spp. and common foodborne pathogens to ensure discriminatory accuracy. Practical applicability was evaluated by analyzing unprocessed environmental water samples, demonstrating the platform’s potential for real-world applications.
Materials and methods
Materials
The bacterial strains and culture conditions used in this study are listed in Table 1.
Methods
Genomic DNA extraction
The bacterial strains were cultured under the conditions specified in Table 1. Genomic DNA was extracted from 1 mL of bacterial culture using a Bacterial Genomic DNA Extraction Kit (DP302, Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. The purified DNA was dissolved in 50 μL of nuclease-free water and stored at −20°C for subsequent analysis.
Construction of standard plasmid
The full-length DNA sequence of the lasR gene (720 bp, Gene ID: 881789) from P. aeruginosa was synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China) and cloned into the pUC57 vector. The resulting recombinant plasmid, designated as pUC-lasR, was used as a quantification standard.
Design and synthesis of primers and probes
RPA primers targeting the lasR gene were designed using Primer Premier 5.0. The primer set comprised four forward primers (lasR-F1, lasR-F2, lasR-F3, and lasR-F4) and one reverse primer (lasR-R1) synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). Three sgRNA (sgRNA527, sgRNA553, and sgRNA618), an ssDNA probe, and a biotin-labeled probe were designed and synthesized by Synbio Technologies Co. Ltd. (Beijing, China). The nucleotide sequences of all primers, sgRNA, ssDNA, and biotin-labeled probes are provided in Table 2.
RPA-CRISPR/Cas12b-Based Fluorescent (RPA-Cas12b-Fluo) Single-Tube Two-Step Method
The RPA-Cas12b-Fluo method was adapted from the protocol established by An et al. [3] and implemented using a SynSor DNA/RNA Isothermal Rapid Amplification Kit (XS-R-101; Synbio Technologies Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The detection workflow was performed in a single tube, comprising two sequential stages: RPA amplification and CRISPR/Cas12b detection.
The RPA amplification mixture was prepared in the reaction tube by thoroughly resuspending the lyophilized isothermal amplification pellet with 12 µL of Amplification Buffer A, followed by sequential addition of 2 µL forward primer (10 µM), 2 µL reverse primer (10 µM), 1 µL magnesium acetate (350 mM), and 1 µL pUC-lasR plasmid. Concurrently, the CRISPR/Cas12b detection mixture was pre-loaded onto the interior surface of the tube cap, comprising 1 µL AaCas12b enzyme (Beijing Synsorbio Co., Ltd. XS-R-001–1), 2.5 µL 10 × AaCas12b Buffer, 1 µL sgRNA (100 ng/µL), 0.5 µL ssDNA reporter (100 µM), and 5 µL ddH₂O. The tube was gently sealed and subjected to RPA amplification at 42°C for 30 min. Following initial amplification, brief centrifugation combined the CRISPR/Cas12b detection mixture with the RPA amplicon. The reaction was immediately transferred to an isothermal amplification instrument (Gene II, OptiGene, UK) for CRISPR/Cas12b detection at 42°C for 60 min, with fluorescence signals monitored in the FAM channel at 1-min intervals throughout the incubation period.
Optimization of RPA-Cas12b-Fluo Primers
To identify the optimal primer combination for RPA amplification, four forward primers (lasR-F1, lasR-F2, lasR-F3, and lasR-F4) were individually paired with a reverse primer (lasR-R1). Primer concentrations were tested at 1.25, 2.5, 5, and 10 µM to determine the optimal concentration for maximizing amplification efficiency. sgRNA527 was used for CRISPR/Cas12b detection, and fluorescence signals were monitored in the FAM channel at 1-min intervals. Each experiment was repeated three times.
Optimization of RPA-Cas12b-Fluo sgRNA
Primer pairs lasR-F2 and lasR-R1 were selected for RPA amplification based on prior optimization. Three sgRNA variants (sgRNA527, sgRNA553, and sgRNA618) were individually evaluated for CRISPR/Cas12b detection to determine the optimal sgRNA for target recognition. Fluorescence signals were monitored in the FAM channel at 1-minute intervals to assess the performance of each sgRNA variant. Each experiment was repeated three times.
Optimization of RPA-Cas12b-Fluo reaction temperature
To determine the optimal reaction temperature for the RPA-Cas12b-Fluo assay, the primer pair lasR-F2 and lasR-R1 was used for RPA amplification, whereas sgRNA527 was used for CRISPR/Cas12b detection. Reaction temperature optimization was performed across a temperature gradient of 37°C, 38°C, 39°C, 40°C, 41°C, and 42°C. Fluorescence signals were monitored in the FAM channel at 1-min intervals to evaluate the amplification efficiency and specificity under each temperature condition. Each experiment was repeated three times.
RPA-Cas12b-LFS
The RPA-Cas12b-LFS assay was conducted using a SynSor DNA/RNA Isothermal Rapid Amplification Kit (XS-R-101, Synbio Technologies Co., Ltd., Beijing, China). The reaction setup and amplification conditions were identical to those described for the RPA-Cas12b-Fluo method, except that the ssDNA probe was replaced with a biotin-labeled probe. Following amplification, 21.4 µL of ddH2O was added to the PCR reaction tube to adjust the total volume to 50 µL, followed by vortex mixing. A Synsor CRISPR detection LFS (XS-R-102; Synbio Technologies Co., Ltd.) was inserted into the reaction tube and incubated at room temperature for 2–5 min. The results were visually interpreted once the liquid front migrated past the control (C) and test (T) lines, with positive signals indicated by the simultaneous appearance of the C and T lines.
Sensitivity analysis of RPA-CRISPR/Cas12b
To establish a robust fluorescence threshold for distinguishing positive signals from background noise in the RPA-Cas12b-Fluo assay, a sensitivity analysis was performed using negative controls. The fluorescence intensity was measured across eight independent negative control replicates. And the determination of the cut-off threshold was conducted in accordance with the method described by Zhao et al [27].
The sensitivities of the RPA-Cas12b-Fluo and RPA-Cas12b-LFS methods were evaluated using these two approaches.
DNA Sensitivity: Serial dilutions of the pUC-lasR plasmid DNA were prepared at final concentrations of 1.2 × 104, 1.2 × 103, 1.2 × 102, and 1.2 × 101 copies. Each dilution was analyzed using both detection methods to determine the limit of detection (LOD) for plasmid DNA.
P. aeruginosa Sensitivity: Genomic DNA was extracted from 1.0 mL P. aeruginosa cultures with viable cell counts of 200, 100, 50, and 25 CFU/mL, and was finally eluted in 50 μL of nuclease-free water for preservation at −20°C. The extracted DNA samples were subjected to RPA-Cas12b-Fluo and RPA-Cas12b-LFS analyses to evaluate the LOD of P. aeruginosa genomic DNA.
For all sensitivity assays (DNA and bacterial detection), experiments were conducted in biological triplicate (n = 3 independent samples per concentration).
Specificity analysis of RPA-CRISPR/Cas12b
To evaluate the specificity of the RPA-Cas12b-Fluo and RPA-Cas12b-LFS methods, genomic DNA from a panel of bacterial strains was tested. The bacterial strains used were Salmonella Enteritidis, Shigella flexneri, Staphylococcus aureus, Listeria monocytogenes, E. coli, S. typhimurium, Pseudomonas fluorescens, P. putida, and P. aeruginosa. Each strain was cultured under the conditions specified in Table 1, and genomic DNA was extracted as previously described. The extracted DNA samples were subjected to the RPA-Cas12b-Fluo and RPA-Cas12b-LFS assays to validate the specificity of the detection methods. For all specificity assays, each experiment was repeated three times.
Water sample detection
Twenty water samples (10 mL each) were processed to evaluate the practical applicability of the RPA-CRISPR/Cas12b assay. Each sample was centrifuged at 8,000 × g for 5 min, and the supernatant was discarded. The pellet was resuspended in 600 μL of phosphate-buffered saline, transferred to a 2-mL tube, and subjected to a second centrifugation at 12,000 rpm for 2 min. The final pellet was retained for genomic DNA extraction using a Bacterial Genomic DNA Extraction Kit. The extracted DNA was analyzed using the national standard microbial culture identification method and RPA-CRISPR/Cas12b assay to validate the accuracy and reliability of the developed platform. Each experiment was repeated three times.
Statistical analysis
All quantitative experiments were performed with biological replicates (n = 3 independent samples per condition). Statistical analysis was conducted using GraphPad Prism 9.0 (GraphPad Software, USA). For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed, followed by Tukey’s honestly significant difference (HSD) post-hoc test for pairwise comparisons. In the bar graphs, significant differences between groups are denoted by different lowercase letters (e.g., a, b, c). Specifically, groups labeled with the same letter are not statistically different from each other, whereas groups labeled with different letters are significantly different at P < 0.01. Data are presented as the mean ± standard error of the mean (SEM).
Results
Screening of RPA-Cas12b-Fluo primers for P. aeruginosa
Four primer pairs were synthesized and tested using a P. aeruginosa standard plasmid as the template to identify the optimal RPA amplification primers. As shown in Fig 1a, Amplification curves generated during 1 hour of RPA-Cas12b-Fluo analysis indicated that the F2-R1 primer pair exhibited consistently higher fluorescence intensity compared to the other three primer pairs, with no false-positive signals observed. The F2-R1 primer pair exhibited the highest fluorescence intensity (240,000) after 1 h of amplification, while the other three primer pairs showed significantly lower intensities relative to F2-R1 (P < 0.01) (Fig 1b). Consequently, the F2-R1 primer pair was selected for subsequent experiments.
(a) Amplification kinetics of four primer pairs during 1 h RPA-Cas12b-Fluo reactions; (b) Endpoint fluorescence intensities comparison of primers (1 h); (c) Concentration-dependent amplification profiles of F2-R1 primer pair (1.25-10 μM); (d) Endpoint fluorescence comparison of primer concentrations (1 h). Different lowercase letters above bars indicate statistically significant differences between groups (P < 0.01). Data: mean ± SEM (n = 3).
To further optimize primer concentration for RPA amplification, the F2-R1 primer pair was tested at concentrations of 1.25, 2.5, 5, and 10 µM. Throughout the 1-hour RPA-Cas12b-Fluo amplification (Fig 1c), the 10 µM F2-R1 primer pair consistently demonstrated higher fluorescence values than other primer concentrations. The 10 µM F2-R1 primer pair yielded a significantly stronger fluorescence signal than the 1.25 µM and 2.5 µM pairs (P < 0.01), and performed comparably to the 5 µM pair (Fig 1d). Consequently, a primer concentration of 10 µM was used for RPA amplification.
Screening of RPA-Cas12b-Fluo sgRNA for P. aeruginosa detection
To determine the optimal sgRNA for the Cas12b-based detection of P. aeruginosa, three sgRNA variants (sgRNA527, sgRNA553, and sgRNA618) were synthesized and evaluated using the P. aeruginosa standard plasmid as the template. Fluorescence amplification curves (Fig 2a) demonstrated that during 1 h RPA-Cas12b reactions at 42°C, sgRNA553 exhibited higher fluorescence intensity than sgRNA527 and sgRNA618. Following 1 h amplification, sgRNA553 demonstrated superior performance, generating a significantly higher fluorescence intensity compared to the other sgRNA variants (P < 0.01) (Fig 2b). These results indicate that sgRNA553 provides superior target recognition and signal amplification compared with other sgRNA variants. Thus, sgRNA553 was identified as the optimal sgRNA for the Cas12b-based detection of P. aeruginosa.
(a) Amplification kinetics of three sgRNA during 1 h RPA-Cas12b-Fluo reactions; (b) Endpoint fluorescence intensities comparison of sgRNA (1 h). Different lowercase letters above bars indicate statistically significant differences between groups (P < 0.01). Data: mean ± SEM (n = 3).
Optimization of reaction temperature for RPA-Cas12b-Fluo
To determine the optimal reaction temperature for the RPA-CRISPR/Cas12b system, a temperature gradient of 37–42°C in 1°C increments was evaluated. Amplification monitoring (Fig 3a) revealed superior fluorescence at 42°C versus other temperatures during RPA-Cas12b-Fluo. Endpoint quantification confirmed this advantage: 42°C samples reached the highest fluorescence intensity, significantly (P < 0.01) exceeding other temperature conditions(Fig 3b). These results highlight the temperature-dependent performance of the RPA-CRISPR/Cas12b system, with 42°C providing optimal conditions for both amplification efficiency and signal generation. Based on these findings, all subsequent RPA-CRISPR/Cas12b experiments were conducted at 42°C to ensure maximum analytical sensitivity and specificity.
(a) Amplification kinetics of critical temperatures during 1 h RPA-Cas12b-Fluo reactions; (b) Endpoint fluorescence intensities comparison of reaction temperature (1 h). Different lowercase letters above bars indicate statistically significant differences between groups (P < 0.01). Data: mean ± SEM (n = 3).
Cut-off threshold testing of the RPA-CRISPR/Cas12b-Fluo assay
Sensitivity testing was performed using negative controls to establish a fluorescence threshold for defining positive reactions in the RPA-CRISPR/Cas12b assay. As shown in Fig 4, eight negative control experiments were conducted, and all fluorescence intensity values remained consistently below 20,000. Based on these findings, a fluorescence intensity value of 20,000 was established as the cut-off threshold for distinguishing positive reactions from background noise. This threshold ensures the reliable discrimination of target-specific signals while minimizing false-positive results.
Fig 4 illustrates the fluorescence intensity values obtained from eight negative control experiments. All values remained below 20,000, establishing a fluorescence intensity of 20,000 as the threshold for distinguishing positive reactions from the background noise.
Sensitivity analysis of the RPA-CRISPR/Cas12b methods
Sensitivity for DNA Templates. Using the pUC-lasR plasmid as the template, fluorescence intensities exceeding the established threshold of 20,000 were detected at final concentrations of 1.2 × 10⁴, 1.2 × 10³, 1.2 × 10², and 1.2 × 10¹ copies per reaction. Following 60 minutes of CRISPR/Cas12b-Fluo detection, the mean fluorescence values for 1.2 × 104, 1.2 × 10³, 1.2 × 10², and 1.2 × 10¹ copies per reaction were all significantly exceeding the cut-off threshold of 20,000 (P < 0.01) (Fig 5a). Therefore, the LOD for the RPA-Cas12b-Fluo method was determined to be 1.2 × 10¹ copies per reaction. For the RPA-Cas12b-LFS method, a visible positive test band on the LFS was observed when the final concentration of pUC-lasR reached 1.2 × 10² copies per reaction (Fig 5b). These results demonstrate the high sensitivity of both methods, with the RPA-Cas12b-Fluo method achieving a lower LOD than the RPA-Cas12b-LFS method.
(a) Sensitivity analysis of RPA-Cas12b-Fluo on DNA template. Different lowercase letters above bars indicate statistically significant differences between groups (P < 0.01). Data: mean ± SEM (n = 3); (b) Sensitivity analysis of RPA-Cas12b-LFS on DNA template.
Sensitivity for P. aeruginosa Cells. To evaluate the sensitivity of the RPA-Cas12b-Fluo and RPA-Cas12b-LFS methods for detecting P. aeruginosa, genomic DNA was extracted from bacterial cultures with viable counts of 200, 100, 50, and 25 CFU/mL. The extracted DNA was analyzed using both methods. Results indicated that the RPA-Cas12b-Fluo method exhibited fluorescence intensities exceeding the established threshold of 20,000 at bacterial loads as low as 50 CFU/reaction, enabling a clear classification as a positive result (Fig 6a). In contrast, the RPA-Cas12b-LFS method demonstrated an LOD of 200 CFU/reaction with no visible test bands on the LFS at bacterial concentrations below 200 CFU (Fig 6b). These findings highlighted the superior sensitivity of the RPA-Cas12b-Fluo method for detecting low concentrations of P. aeruginosa.
(a) Sensitivity analysis of RPA-Cas12b-Fluo on P. aeruginosa. Different lowercase letters above bars indicate statistically significant differences between groups (P < 0.01). Data: mean ± SEM (n = 3); (b) Sensitivity analysis of RPA-Cas12b-LFS on P. aeruginosa.
Specificity analysis of the RPA-CRISPR/Cas12b system
To evaluate the specificity of the RPA-CRISPR/Cas12b system, genomic DNA from three Pseudomonas species (P. aeruginosa, P. fluorescens, and P. putida) and six foodborne pathogens (Salmonella enteritidis, Salmonella typhimurium, Shigella flexneri, Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli) were analyzed. The results are shown in Fig 7. After a 1-h RPA-CRISPR/Cas12b reaction, the RPA-Cas12b-Fluo method generated a fluorescence signal for P. aeruginosa that was significantly higher than that of all other tested strains. Non-P. aeruginosa strains exhibited no detectable fluorescence signals, and their values were not significantly different from those of the template-free control (Fig 7a). Consistent results were obtained using the RPA-Cas12b-LFS method, in which only P. aeruginosa produced a visible test band on the LFS (Fig 7b). These findings demonstrate that the RPA-CRISPR/Cas12b system exhibits high specificity for P. aeruginosa with no detectable cross-reactivity against other pathogenic microorganisms, confirming its potential for accurate and reliable detection in complex samples.
(a) Specificity analysis of RPA-Cas12b-Fluo; (b) Specificity analysis of RPA-Cas12b-LFS.
Application of RPA-CRISPR/Cas12b in water sample analysis
To assess the practical utility of the developed RPA-Cas12b-Fluo system for environmental monitoring, commercially available purified water samples were spiked with varying concentrations of P. aeruginosa and analyzed using the established assay. As summarized in Table 3, both the conventional microbiological culture-based identification method and the RPA-CRISPR/Cas12b assay identified 15 positive and 5 negative samples, demonstrating complete concordance between the two approaches. These results validated the reliability and accuracy of the RPA-CRISPR/Cas12b system for detecting P. aeruginosa in water samples, highlighting its potential as a rapid and effective alternative to traditional culture-based methods for environmental surveillance.
Discussion
In recent years, the integration of RPA with the CRISPR/Cas12b technology has emerged as a powerful tool for the rapid detection of various pathogens and antimicrobial resistance genes. This approach has been successfully used to identify S. enterica [22], resistance genes such as mcr-1 and tet(X4) [28], and SARS-CoV-2 [29]. CRISPR/Cas12b has been used to detect diverse pathogenic bacteria, including Mycobacterium tuberculosis [30], Vibrio parahaemolyticus [31], and Salmonella spp. [32]. Despite these advances, few studies have reported the combination of RPA and CRISPR/Cas12b for the rapid detection of P. aeruginosa. This study establishes the first RPA-CRISPR/Cas12b platform for detecting P. aeruginosa, targeting the previously unexplored lasR gene. Unlike existing Cas12a-based methods requiring temperature switching [23,25], our single-tube, two-step workflow maintains a uniform 42°C isothermal condition, eliminating the need for specialized equipment. This design reduces contamination risks and operational complexity, making it ideal for resource-limited settings.
Regarding target gene selection, no studies have reported an RPA-CRISPR-integrated detection method targeting the lasR gene of P. aeruginosa. Tian et al. [33] developed a graphene oxide-fluorescence-based biosensor for detecting the lasR gene in P. aeruginosa, while Nair [34] designed CRISPR-Cas9-mediated guide RNAs targeting lasR to knock out the gene, thereby reducing biofilm formation and virulence gene expression in P. aeruginosa. These studies collectively validate the feasibility of targeting the lasR gene for P. aeruginosa detection.
In 2023, Liu et al. [25] developed an RPA-CRISPR/Cas12a assay targeting the oprL gene of P. aeruginosa, achieving a genomic DNA detection sensitivity of 60 fg (approximately eight copies) with an agarose gel electrophoresis detection limit of 6 pg. Similarly, Zhang et al. [23] established an RPA-CRISPR/Cas12a system targeting the lasB gene, demonstrating high sensitivity for recombinant plasmid detection, with an LOD of 100 copies/µL via fluorescence analysis and 10¹ copies/reaction using the LFS method. Furthermore, Yang et al. [12] developed a single-tube RPA-CRISPR/Cas12a assay targeting the lasB gene of P. aeruginosa, achieving a bacterial cell sensitivity of 15.9 CFU/reaction and a 97.62% concordance rate with conventional clinical detection methods. Our platform achieved a sensitivity of 10 DNA copies/reaction (fluorescence) and 50 CFU/reaction, comparable to Cas12a-based assays [23,25] and with enhanced workflow simplicity. While the LFS sensitivity (200 CFU) was lower than Yang et al.’s Cas12a-LFS (15.9 CFU) [12], our method requires no intermediate amplicon transfer, significantly reducing false positives. The lasR-targeting system demonstrated 100% specificity against related Pseudomonas spp. and foodborne pathogens, outperforming oprL/lasB-based assays in discriminatory accuracy [23,25].
Notwithstanding its high analytical sensitivity, this platform exhibits several limitations: restricted matrix diversity currently confined to aqueous samples; uncharacterized susceptibility to matrix interference from complex backgrounds such as food or clinical specimens; and reduced sensitivity in LFS format relative to fluorescence detection. Future validation studies will assess platform performance in clinical matrices while integrating portable readers to enable quantitative LFS analysis. Compared to traditional methods for P. aeruginosa detection, our RPA-CRISPR/Cas12b platform offers distinct advantages. Standard culture-based methods, as stipulated in protocols like China’s GB 8537−2018 standard for bottled drinking water, although reliable for determining bacterial viability and serving as the regulatory gold standard, typically require 24–72 hours for completion and involve labor-intensive steps of selective culturing and biochemical confirmation. Molecular techniques like PCR and quantitative PCR (qPCR) offer improved speed and specificity but remain reliant on thermal cyclers and sophisticated laboratory infrastructure, limiting their use in field or point-of-care settings [10]. In contrast, our single-tube, isothermal platform completes detection within approximately 90 minutes, requires only a simple heating block or portable incubator, and reduces the risk of amplicon contamination through its closed-tube, two-step design. The integration with LFS further simplifies result interpretation, making it suitable for non-laboratory environments. However, it is important to note that traditional culture methods retain the unique advantage of providing information on bacterial viability and enabling antimicrobial susceptibility testing, which are crucial in clinical management.
Conclusion
We successfully developed a single-tube, two-step RPA-CRISPR/Cas12b platform targeting the lasR gene of P. aeruginosa, which enabled rapid, sensitive, and specific pathogen detection. Through systematic optimization of the reaction conditions, primer pairs, and sgRNA variants, the optimal primer pair (F2-R1) and sgRNA (sgRNA553) were identified. Using fluorescence-based analysis, the platform achieved an LOD of 10 copies/reaction for DNA and 50 CFUs/reaction for bacterial cells. Integration with LFS provides a field-friendly option, achieving an LOD of 102 copies/reaction and 200 CFU/reaction. The assay demonstrated exceptional specificity in distinguishing P. aeruginosa from closely related Pseudomonas spp. and common foodborne pathogens without cross-reactivity. Validation using 20 water samples showed 100% concordance with the national standard culture methods, confirming its reliability and practical applicability in real-world scenarios. This platform offers a versatile solution for laboratory and field applications owing to its high sensitivity, specificity, and operational simplicity.
Supporting information
S1 Raw Date.
Supporting information-Raw Date.
https://doi.org/10.1371/journal.pone.0340856.s001
(XLSX)
References
- 1. Krell T, Matilla MA. Pseudomonas aeruginosa. Trends Microbiol. 2024;32(2):216–8. pmid:38065787
- 2. Tang Y, Ali Z, Zou J, Jin G, Zhu J, Yang J, et al. Detection methods for Pseudomonas aeruginosa: history and future perspective. RSC Adv. 2017;7(82):51789–800.
- 3. An B, Zhang H, Su X, Guo Y, Wu T, Ge Y, et al. Rapid and Sensitive Detection of Salmonella spp. Using CRISPR-Cas13a Combined With Recombinase Polymerase Amplification. Front Microbiol. 2021;12:732426. pmid:34733250
- 4. Miao J, Zuo L, He D, Fang Z, Berthet N, Yu C, et al. Rapid detection of Nipah virus using the one-pot RPA-CRISPR/Cas13a assay. Virus Res. 2023;332:199130. pmid:37178792
- 5. Yao C, Zhang R, Tang J, Yang D. Rolling circle amplification (RCA)-based DNA hydrogel. Nat Protoc. 2021;16(12):5460–83. pmid:34716450
- 6. Lai MY, Sohairi AN, Zen LPY, Abdullah ML, Lau YL. Loop-Mediated Isothermal Amplification for Diagnosis of Zoonotic Malaria. Am J Trop Med Hyg. 2024;111(4):765–9. pmid:39106849
- 7. Xiang Y, YaN L, Zheng X, Li L, Liu P, Cao W. Rapid detection of Pseudomonas aeruginosa by cross priming amplification. Journal of Integrative Agriculture. 2020;19(10):2523–9.
- 8. Ma B, Li J, Chen K, Yu X, Sun C, Zhang M. Multiplex Recombinase Polymerase Amplification Assay for the Simultaneous Detection of Three Foodborne Pathogens in Seafood. Foods. 2020;9(3):278. pmid:32138267
- 9. Jin XJ, Gong YL, Yang L, Mo BH, Peng YZ, He P, et al. Application of recombinase polymerase amplification in the detection of Pseudomonas aeruginosa. Zhonghua Shao Shang Za Zhi. 2018;34(4):233–9. pmid:29690742
- 10. Tran DH, Tran HT, Pham TNM, Phung HTT. Direct multiplex recombinase polymerase amplification for rapid detection of Staphylococcus aureus and Pseudomonasaeruginosa in food. Mol Biol Res Commun. 2022;11(1):1–10. pmid:35463821
- 11. Xu Y, Wu Y, Liang L, Huang P, Chen R, Qin C, et al. Real-Time Recombinase Polymerase Amplification (RPA) Detection of Pseudomonas aeruginosa Using Magnetic Nano-Beads for DNA Extraction. sci adv mater. 2021;13(9):1657–65.
- 12. Yang H, Wang Y, Yang Q, Fan H, Wang L, Zhang T, et al. A Rapid and Sensitive Detection Method for Pseudomonas aeruginosa Using Visualized Recombinase Polymerase Amplification and Lateral Flow Strip Technology. Front Cell Infect Microbiol. 2021;11:698929. pmid:34595129
- 13. Zhou J, Yin L, Dong Y, Peng L, Liu G, Man S, et al. CRISPR-Cas13a based bacterial detection platform: Sensing pathogen Staphylococcus aureus in food samples. Anal Chim Acta. 2020;1127:225–33. pmid:32800128
- 14. Chen X, Huang C, Zhang J, Hu Q, Wang D, You Q, et al. Mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) and its application in RNA detection. Talanta. 2024;268(Pt 1):125350. pmid:37922816
- 15. Zhang Y, Liu H, Nakagawa Y, Nagasaka Y, Ding T, Tang S-Y, et al. Enhanced CRISPR/Cas12a-based quantitative detection of nucleic acids using double emulsion droplets. Biosens Bioelectron. 2024;257:116339. pmid:38688231
- 16. Hao W, Cui W, Liu Z, Suo F, Wu Y, Han L, et al. A New-Generation Base Editor with an Expanded Editing Window for Microbial Cell Evolution In Vivo Based on CRISPR‒Cas12b Engineering. Adv Sci (Weinh). 2024;11(22):e2309767. pmid:38602436
- 17. Yang H, Gao P, Rajashankar KR, Patel DJ. PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease. Cell. 2016;167(7):1814-1828.e12. pmid:27984729
- 18. Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using recombination proteins. PLoS Biol. 2006;4(7):e204. pmid:16756388
- 19. Teng F, Guo L, Cui T, Wang X-G, Xu K, Gao Q, et al. CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity. Genome Biol. 2019;20(1):132. pmid:31262344
- 20. Li L, Li S, Wu N, Wu J, Wang G, Zhao G, et al. HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation. ACS Synth Biol. 2019;8(10):2228–37. pmid:31532637
- 21. Yang L, Chen G, Wu J, Wei W, Peng C, Ding L, et al. A PAM-Free One-Step Asymmetric RPA and CRISPR/Cas12b Combined Assay (OAR-CRISPR) for Rapid and Ultrasensitive DNA Detection. Anal Chem. 2024;96(14):5471–7. pmid:38551977
- 22. Gong J, Zhang D, Fu L, Dong Y, Wu K, Dou X, et al. A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection. Microorganisms. 2024;12(3):519. pmid:38543570
- 23. Zhang W, Qu H, Wu X, Shi J, Wang X. Rapid, sensitive, and user-friendly detection of Pseudomonas aeruginosa using the RPA/CRISPR/Cas12a system. BMC Infect Dis. 2024;24(1):458. pmid:38689239
- 24. Yang H, Liu A, Ma F, Gao X, Wang K, Wang Y. Establishment of portable Pseudomonas aeruginosa detection platform based on one-tube CRISPR/Cas12a combined with recombinase polymerase amplification technology. Clin Chim Acta. 2024;554:117760. pmid:38176521
- 25. Liu S, Huang S, Li F, Sun Y, Fu J, Xiao F, et al. Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system. Front Cell Infect Microbiol. 2023;13:1239269. pmid:37637458
- 26. Zhu X-X, Wang Y-S, Li S-J, Peng R-Q, Wen X, Peng H, et al. Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification. Front Microbiol. 2024;15:1341179. pmid:38357344
- 27. Zhao Y, Dai J, Zhang Z, Chen J, Chen Y, Hu C, et al. CRISPR-Cas13a-Based Lateral Flow Assay for Detection of Bovine Leukemia Virus. Animals (Basel). 2024;14(22):3262. pmid:39595314
- 28. Wang Y, Chen H, Pan Q, Wang J, Jiao X, Zhang Y. Development and evaluation of rapid and accurate one-tube RPA-CRISPR-Cas12b-based detection of mcr-1 and tet(X4). Appl Microbiol Biotechnol. 2024;108(1):345. pmid:38801527
- 29. Aman R, Marsic T, Sivakrishna Rao G, Mahas A, Ali Z, Alsanea M, et al. iSCAN-V2: A One-Pot RT-RPA-CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection. Front Bioeng Biotechnol. 2022;9:800104. pmid:35127671
- 30. Sam IK, Chen Y-Y, Ma J, Li S-Y, Ying R-Y, Li L-X, et al. TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis. J Infect. 2021;83(1):54–60. pmid:33951419
- 31. Wang S, Gao Y, Miao Y, Jiang W, Zhang X, Zheng Y, et al. Detection of Vibrio parahaemolyticus by one-pot LAMP-CRISPR/Cas12b combined with immunomagnetic beads. Food Control. 2025;172:111181.
- 32. Zhang C, Luo Z, Lin X. One-pot dual-CRISPR/Cas12b-mediated dUTP-LAMP for rapid and sensitive detection of foodborne pathogens without carryover contamination. Food Bioscience. 2024;59:104069.
- 33. Tian L, Chen S, Ning Y. Monitoring of lasR gene transcription in Pseudomonas aeruginosa via a graphene oxide-based fluorometric bioassay coupling strand displacement-triggered target recycling and exonuclease III-assisted signal amplification. Microchemical Journal. 2025;208:112617.
- 34. Radha KesavanNair L. Computational design of guide RNAs and vector to knockout LasR gene of Pseudomonas aeruginosa. Gene and Genome Editing. 2023;6:100028.