Rapid, sensitive, and user-friendly detection of Pseudomonas aeruginosa using the RPA/CRISPR/Cas12a system

The Isothermal DNA Amplification Basic Kit for RPA was sourced from EZassay Ltd. (Shenzhen, China). LbaCas12a enzyme and its corresponding reaction buffer were purchased from New England Biolabs (Ipswich, MA, USA). The TIANamp Bacteria DNA kit was sourced from Tiangen Biotech Co., Ltd (Beijing, China). Ultrapure water (dd H₂O) was purchased from Thermo Fisher Scientific (Shanghai, China). RPA primers, crRNA and the ssDNA reporters (fluorescence assay: 5′ FAM - TTATT - BHQ1 3′, lateral flow test strip (LFTS) method: 5′ FITC - TTTTTTTTTT - Biotin 3′) were custom-synthesized by Sangon Biotech Co.,Ltd (Shanghai, China). The ABI 7500 fast system was purchased from Applied Biosystems (Foster City, CA, United States). Lateral flow strips were obtained from Tiosbio (Beijing, China) and their principle is illustrated in Fig. 1. The test strip’s conjugate pad was coated with complexes of gold nanoparticles and anti-FITC antibody. Streptavidin (SA) and goat anti-mouse IgG were immobilized on the NC membrane at the positions corresponding of the C line and the T line, respectively (Fig. 1B). In the absence of the target, the trans-cleavage activity of Cas12a remained inactive, keeping the ssDNA reporters intact (Fig. 1A). Complete ssDNA reporters, along with complexes of gold nanoparticle and anti-FITC antibody, were captured by SA on the C line, resulting in the visibility of the C line while the T line invisible (Fig. 1C). In the presence of the target, the trans-cleavage activity of Cas12a was activated, leading to the separation of FITC and biotin molecules within the ssDNA reporters. FITC-anti-FITC antibody-gold nanoparticle complexes were captured by the goat anti-mouse IgG on the T line, rendering the T line visible, with no change to the visibility of the C line (Fig. 1D).

A standard P. aeruginosa strain, four clinically isolated P. aeruginosa strains, and ten non - P. aeruginosa strains were generously provided by Autobio Diagnostics Co., Ltd. Detailed information on the strains used in this study is presented in Table 1. The bacterial strains were stored in Luria-Bertani (LB) broth supplemented with 20% glycerol at -70℃. When required, the preserved bacteria were streaked onto agar plates and incubated at 37℃ for 24 h. Single colonies were picked and further incubated at 37℃ for 24 h. The bacterial suspension was subsequently standardized to a final concentration of 1 × 10⁵ CFU/mL for subsequent experimental procedures. Genomic DNA was extracted from these bacterial cultures using the TIANamp Bacteria DNA kit for all experiments except sensitivity analysis, following the standard manufacturer’s instructions.

For the detection of P. aeruginosa, the lasB gene was selected as the target due to its significant intraspecific conservation and notable interspecific similarity, as observed in the genome sequences of P. aeruginosa from GenBank. To ensure specificity, RPA primers and crRNA were meticulously designed to target specific regions within the lasB gene. The design locations for the RPA primers and crRNA are visually represented in Fig. 2, and the sequences of these primers and crRNA can be found in Table 2.

The RPA amplification was conducted using the Isothermal DNA Amplification Basic Kit following the manufacturer’s standard instructions. The amplification system (20µL) comprised the following components: Rehydration Buffer (2X) 10µL, Forward Primer (20µM) 0.5µL, Reverse Primer (20µM) 0.5µL, DNA template 1µL, ddH₂O 6µL, and Starter (10X) 2µL. The mixture was gently mixed by flicking and centrifuged at low speed for 10 s, repeat this step three times. The reaction system was then incubated at 39℃ for various duration, including 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, to determine the optimal incubation time. As a negative control, ddH₂O was used.

To identify the crRNA for CRISPR/Cas12a detection, crRNA1 and crRNA2 were separately introduced into the CRISPR/Cas12a detection reaction system, followed by the observation of fluorescence intensity. The reaction system (40µL) consisted of the following components: 10µL reaction buffer (4X), 1µL Reporter (4µM, 5′ FAM – TTATT - BHQ1 3′), 2µL Cas12a Protein (1µM), 2µL crRNA (1µM), and 23µL ddH₂O, pre-mixed accordingly. Subsequently, 2µL of RPA amplification products were added and thoroughly mixed. The reaction tube was immediately transferred to a Real-time fluorescence PCR instrument. The fluorescence resulting from the Cas12a cleavage reaction was continuously collected using the ABI fast 7500 system. Background-subtracted fluorescence was determined by subtracting the background fluorescence (without template) from the initial fluorescence of all samples. Subsequently, the background subtracted fluorescence intensities were compared under the same reaction conditions to select the optimal crRNA and reaction time based on the results obtained.

The specificity of the RPA/CRISPR/Cas12a detection platform was assessed using a range of strains as listed in Table 1. This included 1 standard strain of P. aeruginosa, 4 clinically isolated of P. aeruginosa, 4 Pseudomonas strains closely related to P. aeruginosa, and 6 non-Pseudomonas strains. Each strain was individually subjected to detection using the RPA/CRISPR/Cas12a platform at a concentration of 1 × 10⁵ CFU/mL. The assays were performed three times, following the methodology described above.

To assess the sensitivity of RPA/CRISPR/Cas12a detection platform, a recombinant plasmid containing the lasB target sequence (pGEM - T Easy - lasB) was subjected to continuous 10 - fold dilution, ranging from 10⁶ to 10⁰ copies/µL. Subsequently, the differently diluted DNA samples were used as templates to determine LOD. All assays were replicated 3 times using the aforementioned methodology.

To validate the practical applicability of the RPA/CRISPR/Cas12a detection platform, a total of 150 diverse food samples were analyzed, including processed meat products, cold seasoned vegetable dishes from nearby markets, and college-provided bottled water. Specific methods for sample processing are referenced in the literature [26]. Additionally, for a comprehensive comparison, these samples were concurrently subjected to detection using qPCR. In particular, qPCR was executed in a 25µL volume with 12.5µL of 2×SYBR Green PCR mix, 2µL of each primer at 1 µM concentration, 1µL of DNA template, and 7.5µL ddH₂O. The PCR program began with a 10-minute denaturation at 95℃, followed by 40 cycles at 95℃ for 15 s for denaturation, and annealing/extension at 60℃ for 30 s, using primers described in prior literature [21]. Each assay was performed in triplicate, adhering strictly to the described protocol.

Statistical analyses were performed using SPSS 21.0 (IBM SPSS 21.0, SPSS, Inc., Chicago, IL, USA). A Studentʼs t test was employed for the comparisons between two groups. All data were presented as mean ± SD. Statistical significance was set as P < 0.05. Each experiment was performed in triplicate.

The RPA product, amplified at the optimal reaction time of 15 min, was employed as the template for screening crRNA in the CRISPR/Cas12a detection. crRNA1 and crRNA2 were separately introduced into the reaction system, and the background-subtracted fluorescence values were compared between the crRNA1 and the crRNA2 groups. As illustrated in Fig. 3, the fluorescence values of the crRNA1 group were higher than those of the crRNA2 group (P < 0.05), establishing crRNA1 as the optimal choice for CRISPR/Cas12a detection. Additionally, Fig. 3 demonstrates that the fluorescence values of the crRNA1 group were consistently higher than the crRNA2 group throughout the duration of the reaction. The fluorescence curve analysis further revealed that background-subtracted fluorescence reached a plateau after approximately 15 min, confirming 15 min as the optimal reaction time for the CRISPR/Cas12a detection method.

The RPA/CRISPR/Cas12a detection platform was employed to assess 15 bacteria strains (Table 1). The fluorescence assay results demonstrated that there was a significant increase in fluorescence values for the P. aeruginosa standard strain and four clinically isolated strains of P. aeruginosa (P<0.05), while fluorescence values remained relatively unchanged for other non-P. aeruginosa strains (Fig. 4A). The outcomes from the LFTS method were consistent with those of the fluorescence assay, showing positive results exclusively for the P. aeruginosa standard strain and 4 P. aeruginosa clinically isolated strains (Fig. 4B). These results confirmed the high specificity of the RPA/CRISPR/Cas12a detection platform in accurately identifying P. aeruginosa.

To assess the sensitivity of the RPA/CRISPR/Cas12a detection platform, a recombinant plasmid containing the lasB target sequence (pGEM - T Easy - lasB) was subjected to serial 10-fold dilutions, ranging from 10⁶ to 10⁰ copies/µL. Each diluted sample was analyzed using the RPA/CRISPR/Cas12a detection platform to determine the LOD, with ddH₂O serving as the negative control. As depicted in Fig. 5, a significant fluorescence signal was observed using fluorescence assay when the DNA concentration exceeded 10⁰ copies/µL (P<0.05). Interestingly, the results obtained from the LFTS method were generally consistent with those of the fluorescence assay.The appearance of T line was observed at a concentration of 10¹ copies/µL and above. Among the positive results, both the T line and C line became visible at a concentration of 10¹ copies/µL, it is speculated that the trans-cleavage activity of Cas12a was insufficiently activated due to the low DNA concentration, resulting in only a portion of ssDNA reporters were trans-cleaved. Consequently, the ssDNA reporter-gold nanoparticle-anti-FITC antibody complexes were captured on the C line, while the FITC-anti-FITC antibody-gold nanoparticle complexes were captured on the T line, making both the T line and C line visible. These results indicate that RPA/CRISPR/Cas12a detection platform exhibits high sensitivity, with a LOD of 10⁰ copies/µL for fluorescence assay and 10¹ copies/µL for the LFTS method.

To evaluate the applicability of RPA/CRISPR/Cas12a detection platform, a total of 150 samples were tested using both qPCR and the RPA/CRISPR/Cas12a detection platform. The results obtained from the fluorescence assay and the LFTS method were compared with the results of the qPCR assay. The findings are presented in Table 3, demonstrating that the results of both the fluorescence assay and the LFTS method were generally consistent with the qPCR assay. This suggests that the performance of the RPA/CRISPR/Cas12a detection platform is comparable to that of qPCR. One notable advantage of the RPA/CRISPR/Cas12a detection platform is its ability to deliver results in a shorter time and at isothermal temperature compared to qPCR. Consequently, the RPA/CRISPR/Cas12a detection platform offers a convenient and efficient alternative for pathogen detection.