What this is
- is a zoonotic disease that poses significant public health challenges due to complex diagnosis.
- This research developed a rapid detection method combining recombinase polymerase amplification () with .
- The assay showed high sensitivity and specificity, with clinical validation confirming its reliability using serum samples.
Essence
- The - assay enables rapid and sensitive detection of Brucella melitensis, achieving a detection limit of 1 copy/μL. This method is suitable for clinical applications, providing a reliable tool for diagnosis.
Key takeaways
- The assay achieved a detection limit of 1 copy/μL using fluorescence, which is 10 times more sensitive than qPCR. This level of sensitivity is crucial for early diagnosis and effective treatment of .
- Clinical validation demonstrated 100% concordance with serological results from 24 patients and six healthy controls. This confirms the assay's reliability for clinical applications.
- The assay's specificity was validated by testing against various bacterial species, showing strong signals only for Brucella, indicating its robustness in distinguishing target pathogens.
Caveats
- The assay was validated with a limited number of clinical samples, necessitating further studies with larger cohorts to confirm its reliability across different populations.
- Performance in detecting other Brucella species remains untested, which could limit its application in diverse epidemiological contexts.
- While the lateral flow strip format offers convenience, its sensitivity is lower than the fluorescence method, suggesting a need for optimization.
Definitions
- Brucellosis: A zoonotic disease caused by Brucella species, often leading to flu-like symptoms and potential chronic complications.
- RPA: Recombinase polymerase amplification, a nucleic acid amplification technique that allows rapid DNA amplification without thermal cycling.
- CRISPR/Cas12a: A bacterial-derived nuclease used for precise nucleic acid detection, known for its high specificity and sensitivity.
Simplified
INTRODUCTION
Brucellosis is an easily overlooked zoonotic disease caused by gram-negative, facultative intracellular Brucella species (1, 2). It is recognized as one of the most widespread zoonotic bacterial infections globally, with the primary strains associated with human infections being B. melitensis, B. abortus, B. suis, and B. canis, among which B. melitensis is the most prevalent and pathogenic (1, 3, 4). Brucella is usually transmitted to humans through consumption of unpasteurized dairy products or direct contact with infected animals and their secretions (5, 6). Clinically, the disease often manifests with acute flu-like symptoms, including fever, chills, malaise, and fatigue, and may progress to a chronic condition (7). It can also result in various complications, such as undulant fever, arthritis, spondylitis, endocarditis, and chronic fatigue syndrome (8, 9). Due to the non-specific nature of the symptoms, human brucellosis is frequently misdiagnosed as other diseases; thus, accurate diagnosis and treatment are crucial for effective management (10).
Commonly used methods for detecting Brucella include bacterial isolation and identification, immunological detection, and nucleic acid detection (11). Of these, the traditional isolation and culture methods, although considered the gold standard, require specialized laboratory conditions and long incubation times to obtain results (12). In addition, the sensitivity of blood cultures may be significantly reduced in cases of chronic diseases and localized infections (12, 13). As a result, immunodiagnostic methods have become more widely used, particularly the Rose Bengal test, the standard tube agglutination test, and the enzyme-linked immunosorbent assay (11). Despite the widespread use of these tests and their rapid results, they have certain limitations. For example, there is a window period for the test, and false-positive results may also occur due to cross-reactivity with other gram-negative bacterial infections, raising concerns about specificity (14, 15).
In recent years, molecular biology methods, such as qualitative and quantitative polymerase chain reaction (qPCR) targeting specific genes, have been widely used for the diagnosis of the disease (16, 17). PCR is a highly sensitive and practical method, capable of amplifying nucleic acids to detect infecting microorganisms in a sample (18). Although PCR-based technology can be used for the diagnosis of brucellosis, its application in resource-poor and high-prevalence areas is limited due to the need for expensive equipment and specialized laboratories. Recombinase polymerase amplification (RPA) is a novel, thermostable nucleic acid amplification technique that provides rapid and efficient amplification of DNA or RNA without the need for complex equipment, making it suitable for field applications and resource-poor environments (19, 20). Previous studies have shown that RPA can effectively detect the Brucella bcsp31, bp26, and IS711 genes with excellent sensitivity and specificity (21, 22). However, RPA has some limitations: for example, RPA may produce non-specific amplification, leading to false positives (23).
The CRISPR/Cas system, especially Cas12a, plays a significant role in the field of molecular diagnosis. Cas12a is a bacterial-derived nuclease that binds to a TTTV protospacer adjacent motif (V = A/G/C) upstream of the target DNA and exhibits non-specific cleavage activity, making it suitable for rapid nucleic acid testing (24). The combination of RPA and Cas12a provides higher sensitivity and specificity than Cas12a alone, and RPA can rapidly amplify the target DNA under isothermal conditions, increasing the sensitivity of the Cas12a assay and enabling efficient detection of pathogens at very low levels of pathogenic nucleic acids (25). This method has been widely used for the rapid diagnosis of pathogens such as SARS-CoV-2 (26), Mycobacterium tuberculosis (27), and Plasmodium falciparum (28), and is suitable for resource-limited areas.
In this study, we designed RPA primers and crRNAs for the B. melitensis omp31 gene as a target sequence and developed a corresponding RPA-CRISPR/Cas12a detection system. We developed an RPA-CRISPR/Cas12a assay by combining RPA and CRISPR/Cas12a in a single-tube reaction system. The assay can be performed using either real-time quantitative fluorescence (FL) analysis or lateral flow immunoassay strips, each of which uses a specific ssDNA reporter molecule for signal readout. The specificity of the method was evaluated by testing a variety of other bacteria, and sensitivity was assessed at the level of molecular detection. In addition, the method was applied to serum samples from patients with Brucella infections in different hospitals in Hangzhou, Zhejiang Province, China, which validated its potential application in clinical diagnosis. In conclusion, this study provides a novel molecular diagnostic strategy for the rapid and sensitive detection of Brucella and lays the foundation for further optimization of clinical detection methods.
RESULTS
Establishment and optimization of RPA-CRISPR/Cas12a one-tube assay
In this study, we developed a rapid detection method for Brucella infections that combines RPA with the CRISPR/Cas12a system, and the final results can be presented by fluorescent or lateral flow strip (LFS), as shown in Fig. 1. In order to improve the detection sensitivity, crRNA screening, primer screening, and optimization of experimental conditions were systematically carried out in our study. Firstly, the whole genome of Brucella was used as a template, and the omp31 gene (Fig. 2A) was amplified by PCR using primers omp31-F and omp31-R (Table S1), and the plasmid PMD-19T-omp31 was obtained by cloning the gene into the pMD-19T vector. Subsequently, for the omp31 gene, three sets of crRNAs were designed in this study (Table S1), and their performance was compared by evaluating the FL intensity of the reaction. After comparison, crRNA1 was determined to be the best choice for brucellosis detection (Fig. 2B). Regarding RPA amplification primer screening, four pairs of primers were designed and evaluated in this study (Table S1), and the optimal primer combination, F3R3, was finally determined (Fig. 2C). By optimizing the concentrations of Cas12a protein and crRNA through the checkerboard method, we finally determined that the best detection results were achieved at a protein concentration of 1 µM and a crRNA concentration of 0.8 µM (Fig. 3A). In addition, the optimal concentration of the probe was also explored in this study, and after a series of dilution experiments, the probe concentration was finally determined to be 10 µM (Fig. 3B).
The diagram ofdetection based on RPA-CRISPR/Cas12a-FL and RPA-CRISPR/Cas12a-LFS. Genomic DNA extracted from the serum is amplified by the RPA. In the case of a positive sample, crRNA binds to Cas12a to specifically identify the target gene. Subsequently, Cas12a can nonspecifically cut FAM-BHQ1 and FITC-biotin reporter genes. The separation of FAM from BHQ1 enables FL detection by qPCR instrument. In addition, the separation of FITC from biotin makes the Au-nanoparticle anti-FITC bound to it unable to be fully captured by streptavidin in the quality control line, thus forming a visible band on the detection line. B. melitensis
The screening of the RPA-CRISPR/Cas12a assay crRNA and primers. () PCR amplification of theomp31 gene. M, DNA marker; 1–2, omp31 gene; and N, negative control. () Comparison of different crRNAs targeting omp31 by FL assay, N, negative control. () Screening results for RPA primers. A B C B. melitensis
Optimization results of the RPA-CRISPR/Cas12a assay. () Optimization of the concentration of Cas12a and crRNA. () Optimization of the concentration of reporter. A B
RPA-CRISPR/Cas12a assay has robust sensitivity
We used different concentrations of pMD-19T-omp31 plasmid for RPA-CRISPR/Cas12a sensitivity analysis. Statistical analysis through an independent student’s t-test showed that the FL intensity of plasmids with concentrations ranging from 1 × 106 to 1 × 101 copies/μL was significantly higher than that of the negative control group, and the FL intensity of plasmids with a concentration of 1 × 100 copy/μL was also higher than that of the negative control group. The result showed that the lower limit of detection (LOD) of the RPA-CRISPR/Cas12a-FL method was 1 copy/μL (Fig. 4A). In addition, through LFS detection, we can visually observe that the LOD of the RPA-CRISPR/Cas12a-LFS method was 10 copies/μL (Fig. 4B). Meanwhile, we also performed fluorescent PCR and nested PCR assays (Fig. 4C and D). The results showed that the sensitivity of the RPA-CRISPR/Cas12a-FL assay was 10 times higher than that of qPCR, and the sensitivity of the RPA-CRISPR/Cas12a-LFS assay was comparable to that of nested PCR.
The sensitivity analysis of RPA-CRISPR/Cas12a-FL and RPA-CRISPR/Cas12a-LFS. () Sensitivity result of the RPA-CRISPR/Cas12a-FL assay was performed using serial 10-fold dilutions of pMD-19T-omp31 as a template. The concentration gradients detected were 1 × 10copies/μL, 1 × 10copies/μL, 1 × 10copies/μL, 1 × 10copies/μL, 1 × 10copies/μL, 1 × 10copies/μL, and 1 × 10copy/μL, corresponding to numbers 6 to 0, respectively. NC, negative control. () Sensitivity result of the qPCR. NC, negative control. () Sensitivity result of the RPA-CRISPR/Cas12a-LFS assay and nested PCR was performed using serial 10-fold dilutions of pMD-19T-omp31 as a template. The concentration gradients detected were 1 × 10copies/μL, 1 × 10copies/μL, 1 × 10copies/μL, 1 × 10copies/μL, 1 × 10copies/μL, 1 × 10copies/μL, and 1 × 10copy/μL, corresponding to numbers 1 to 7, respectively. N, negative control. ****< 0.0001; ***< 0.001; **< 0.01; *< 0.1. A B C and D 6 5 4 3 2 1 6 5 4 3 2 1 P P P P
RPA-CRISPR/Cas12a assay has robust specificity
To analyze the specificity of the RPA-CRISPR/Cas12a assay, we used DNA samples from a variety of different bacteria, including Vibrio paraholyticus, Klebsiella pneumoniae, Haemophilus influenzae, V. cholerae, Escherichia coli, and Salmonella. As shown in Fig. 5A, the RPA-CRISPR-Cas12a-FL FL assay showed that only Brucella showed a strong fluorescent signal, whereas none of the other bacteria and negative control samples showed a signal. In addition, the RPA-CRISPR-Cas12a-LFS lateral flow immunoassay showed that the Brucella samples had a clear positive color band, while the other bacterial species did not show any positive color band (Fig. 5B). These results indicate that both methods have excellent detection specificity.
The specificity analysis of RPA-CRISPR/Cas12a-FL and RPA-CRISPR/Cas12a-LFS. () Sensitivity result of the RPA-CRISPR/Cas12a-FL assay was performed using DNA from different bacterial species. () Sensitivity result of the nested PCR was performed using DNA from different bacterial species. 1–7 represent BM, VP, KP, HI, VC, EC, and SAL. N, negative control.< 0.0001. A B ****P
RPA-CRISPR/Cas12a assay detection for serum sample
To validate the feasibility of this assay for clinical application, serum samples were collected from 24 confirmed patients with brucellosis and six healthy controls. All samples were serologically validated. After extracting the sample genomes, fluorescent PCR and RPA-CRISPR/Cas12a assays were performed, and the results are shown in Table 1. The two assays were consistent with the serologic results, indicating the clinical application of the method.
| Detection method | Serum samples (= 30)n | |
|---|---|---|
| Positive | Negative | |
| RPA-CRISPR/Cas12a-FL | 24 | 6 |
| RPA-CRISPR/Cas12a-LFS | 24 | 6 |
| qPCR | 24 | 6 |
DISCUSSION
The development of a rapid and sensitive diagnostic assay for detecting B. melitensis is critical in both clinical and veterinary settings, given the significant public health and economic implications of brucellosis. This study successfully developed a novel detection method based on RPA-CRISPR/Cas12a, which combines the rapidity of RPA with the high specificity of CRISPR/Cas12a technology. This assay provides a promising alternative to traditional methods such as culture-based techniques, serological assays, and conventional PCR.
Traditional diagnostic methods for B. melitensis exhibit several limitations. Culture-based methods, while considered the gold standard are time-consuming, requiring several days to weeks for bacterial growth and identification (11, 29). Additionally, they pose a significant risk to laboratory personnel due to the highly infectious nature of Brucella species (30, 31). On the other hand, serological assays are faster but often suffer from cross-reactivity with other pathogens, leading to false-positive results (32). Conventional PCR and real-time PCR offer improved sensitivity and specificity but require sophisticated equipment and skilled personnel, limiting their use in resource-limited settings (33, 34).
In contrast, our detection method based on RPA-CRISPR/Cas12a effectively addresses many of the limitations mentioned above. The RPA technology can rapidly amplify the target DNA at a constant temperature (37–42°C) without the need for thermal cycling, thereby shortening the amplification time to within 20 min (19, 35). By integrating CRISPR/Cas12a technology, the target sequence can be specifically recognized, and after recognition, it exhibits significant trans-cleavage activity, which cleaves the single-stranded fluorescent reporter molecule, generating a detectable signal (36, 37). This combination of RPA and CRISPR/Cas12a results in a detection method characterized by high sensitivity and strong specificity. The entire detection process can be completed within 1 h, making it particularly suitable for rapid and accurate clinical diagnosis (38, 39).
This study evaluates detection methods based on FL and LFSs, each possessing unique advantages in a diagnostic setting. The sensitivity of the RPA-CRISPR/Cas12a assay was rigorously evaluated using various concentrations of the pMD-19T-omp31 plasmid. The FL-based method (RPA-CRISPR/Cas12a-FL) exhibited a LOD of 1 copy/μL, whereas the LFS method (RPA-CRISPR/Cas12a-LFS) demonstrated an LOD of 10 copies/μL. As shown in Fig. 4B, this sensitivity level is significantly higher than that of conventional qPCR. The enhanced sensitivity of the RPA-CRISPR/Cas12a assay can be attributed to the synergistic combination of RPA, which facilitates rapid and efficient amplification of target DNA at a constant temperature, and the CRISPR/Cas12a system, which offers a highly specific and sensitive detection mechanism through its trans-cleavage activity (24, 40). The specificity of the assay was confirmed by testing against a panel of DNA samples from various bacterial species. The results indicated that only Brucella samples produced a strong fluorescent signal or a positive color band, while no signal was observed for the other bacterial species or negative controls. This high specificity aligns with previous studies utilizing CRISPR-based systems for pathogen detection, underscoring the capability of CRISPR/Cas12a to distinguish target sequences with single-nucleotide precision (41, 42). The absence of cross-reactivity with other pathogens highlights the robustness of the assay and its potential for accurate diagnosis in both clinical and field settings.
The clinical applicability of the RPA-CRISPR/Cas12a assay was validated using serum samples from 24 confirmed brucellosis patients and six healthy controls. The assay results were consistent with serological findings, demonstrating its potential for use in clinical diagnostics. The ability to detect Brucella DNA in serum samples is particularly important, as serum is a commonly used sample type for brucellosis diagnosis (43).
Despite the promising results, this study has several limitations. First, the assay was primarily validated using laboratory-prepared plasmid DNA and a limited number of clinical serum samples. Further validation with a larger cohort of clinical samples, including those from various stages of infection and different geographical regions, is essential to confirm its robustness and reliability in diverse settings. Second, the assay’s performance in detecting other Brucella species, such as B. abortus and B. suis, has yet to be evaluated. Developing a detection method for multiple Brucella species simultaneously can not only improve the detection sensitivity of different Brucella species but also enable rapid diagnosis of multiple Brucella species in epidemic areas, which is conducive to molecular epidemiological investigations and pathogen traceability. Third, while the LFS format offers simplicity and portability, its sensitivity is slightly lower than that of the FL-based method. Future research could focus on optimizing the LFS to improve its sensitivity without compromising ease of use. For example, the reaction time can be appropriately extended, the replacement of other manufacturers of nucleic acid test strips or combined with digital visualization instruments to read the results.
Conclusion
In conclusion, the RPA-CRISPR/Cas12a-based assay developed in this study offers a rapid, sensitive, and specific method for the detection of B. melitensis. The simplicity, speed, and point-of-care testing potential of the assay make it a valuable addition to the existing diagnostics for brucellosis. By addressing the limitations of traditional and molecular methods, the RPA-CRISPR/Cas12a assay has the potential to significantly improve the diagnosis and control of B. melitensis infections, ultimately reducing the burden of brucellosis on public health and agriculture.
MATERIALS AND METHODS
Samples collection and DNA extraction
The B. melitensis, V. paraholyticus, K. pneumoniae, H. influenzae, V. cholerae, E. coli, and Salmonella were stored in our laboratory. All strains were preserved at −70°C using Microbank preservation vials (Pro-Lab Diagnostics, Ontario, Canada) according to the manufacturer’s protocol. A total of 30 serum samples were collected from various hospitals in Hangzhou, Zhejiang Province. Bacterial and serum genomic DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol and stored at −20°C for subsequent use.
Construction of the positive plasmid
The omp31 gene fragment was expanded by PCR reaction using Brucella genome extracted above as a template. The PCR was carried out in a volume of 50 µL containing 25 µL of 2 × HotStart Taq PCR Mix (TIANGEN, Beijing, China), 2 µL of each primer (10 µmol/L), 1 µL genomic DNA template, and 20 µL of sterile distilled water by the conditions with an initial melting step at 94°C for 3 min, followed by 30 cycles with each cycle at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 5 min. Subsequently, we added the A base to the 3′ end of the PCR product using a DNA A-tail Kit (Takara, Kusatsu, Japan). The DNA fragment with the A-tail was then cloned into the pMD19-T vector (Takara). After transformation and resistance plate inoculation, positive clones were screened and sequenced. Finally, the recombinant plasmid pMD-19T-omp31 was obtained and stored at −20°C for subsequent use.
crRNA design and screen
Three crRNAs were designed using conserved and specific Brucella omp31 gene (GenBank: GQ184729↗) as target genes. The specific sequences of crRNA and single-strand probes (ssDNA) were shown in Table S1, which were synthesized by the Sangon Biotech Co., Ltd. (Shanghai, China). In order to screen out the optimal crRNA, we performed a CRISPR/Cas12a assay. In addition, three independent replicates were performed for each set of experiments. The assay system consisted of 1 µL Cas12a protein (New England Biolabs, MA, USA), 1 µL crRNA, 3 µL 10 × NEBuffer 2.0 (New England Biolabs), 1 µL ssDNA probe, 2.5 µL pMD-19T-omp31 gene, and 21.5 µL of sterile distilled water. The reaction tube was placed in the LineGene 9600 Plus instrument (Bioer Tech, Zhejiang, China), and the FL signal was detected at 37°C for 45 min. FL intensity was recorded and used to evaluate the cleavage efficiency of crRNA.
Establishment of RPA-CRISPR/Cas12a one-tube assay
RPA primers were designed with Primer Premier six software (Premier Biosoft, San Francisco, CA, USA) according to the primer design guidelines of the TwistDx company and synthesized by the Sangon Biotech Co., Ltd. Subsequently, the optimal crRNA obtained from the above screening was used to react with these primers, respectively, and finally, the optimal RPA primer was found. The specific reactions were divided into RPA and CRISPR/Cas12a systems. The TwistAmp Basic Kit (TwistDX Cambridge, UK) was used for RPA amplification in a system consisting of 1.2 µL each RPA primer (10 µM), 14.5 µL buffer, 2.5 µL plasmid template, and 1.2 µL MgOAc (280 mM). The CRISPR/Cas12a system consists of 1 µL Cas12a protein, 1 µL crRNA, 1 µL ssDNA probe, and 3 µL 10× NEBuffer 2.0. The RPA system was first added to the PCR tube, and then the CRISPR/Cas12a system was placed on the cap of the PCR tube. After the RPA reaction for 15 min, the two systems were mixed and reacted at 37℃ in a fluorescent PCR instrument for 45 min. Each group of experiments was independently repeated three times. The final FL intensity was recorded to evaluate the optimal RPA primer pair and the feasibility of the reaction.
Optimization of RPA-CRISPR/Cas12a reaction conditions
In single-tube RPA-CRISPR/Cas12a assays, the specific concentration of Cas12a protein and crRNA is critical to obtain the best detection results. In this study, the chessboard method was used to determine the optimal Cas12a and crRNA concentrations (1 µM, 0.8 µM, 0.6 µM, 0.4 µM, and 0.2 µM) by analyzing the FL intensity. In addition, this study also optimized the concentration of the single-strand probe on this basis.
RPA-CRISPR/Cas12a combined with LFS
Based on the above RPA FL detection method, this study also established a lateral flow test strip detection. For the LFS detection, the system is the same as the FL detection method; only the probe is changed to biotin labeling, and the Cas reaction time is adjusted to 25 min. After the reaction, 70 µL PBS buffer was added into the test tube, thoroughly mixed, and vertically inserted into the LFS (Tiosbio, Beijing, China). The results were interpreted within 5–10 min.
Sensitivity and specificity analysis of RPA-CRISPR/Cas12a assay
To determine the detection limit of RPA-CRISPR/Cas12a, the plasmid containing pMD-19T-omp31 was diluted to 1 × 106 copies/μL, 1 × 105 copies/μL, 1 × 104 copies/μL, 1 × 103 copies/μL, 1 × 102 copies/μL, 1 × 101 copies/μL, and 1 × 100 copy/μL, respectively. The sensitivity of RPA-CRISPR-Cas12a FL detection and RPA-CRISPR-Cas12a LFS detection was tested, respectively. The experimental results were compared with those of qPCR and nested PCR. The qPCR was carried out in a volume of 50 µL containing 25 µL of 2× Real-time PCR Master Mix (Toyobo, Osaka, Japan), 1 µL of each primer (Table S1), 2.5 µL of DNA template, and 20.5 µL of sterile distilled water, and amplification was performed on a LineGene 9600 Plus instrument (Bioer Tech). The nested PCR was carried out in a volume of 50 µL containing 25 µL of 2 × HotStart Taq PCR Mix (TIANGEN), 2 µL of each primer (10 µmol/L), 2.5 µL of genomic DNA template, and 18.5 µL of sterile distilled water by the conditions with an initial melting step at 94°C for 3 min, followed by 30 cycles with each cycle at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 5 min. 1 µL of the product of the first round is added to the system of the second round as the template of the second round, and the procedure is the same as the first round. To verify the accuracy of the results, three detections were performed for each concentration of pMD-19T-omp31. In addition, to assess specificity, both methods tested DNA from six non-target bacteria, including V. paraholyticus, K. pneumoniae, H. influenzae, V. cholerae, E. coli, and Salmonella.
Clinical sample testing
The RPA-CRISPR/Cas12a and RPA-CRISPR/ Cas12A-LFS detection methods established in this study were used to detect Brucella in 30 serologically negative or positive samples collected from different hospitals in Hangzhou, Zhejiang Province.