A primer set, reagent kit, and detection method for detecting Brucella.

Abstract

This invention discloses a primer set, kit, and method for detecting Brucella, belonging to the field of gene detection technology. The primer set comprises two pairs of specific RPA primers: primer pairs B1F1 (SEQ ID No. 3) and B1R1 (SEQ ID No. 6) targeting the B1 sequence, and primer pairs B2F1 (SEQ ID No. 8) and B2R1 (SEQ ID No. 11) targeting the B2 sequence. This invention also provides a kit containing this primer set and a detection method based on RPA-CRISPR-Cas12a. This application combines the high efficiency of RPA amplification with the ultra-high specificity of CRISPR-Cas12a detection, overcoming the shortcomings of traditional methods such as long processing time, low sensitivity, and susceptibility to false positives. It achieves rapid, sensitive, and specific detection of Brucella, and has significant application value in the early screening and monitoring of brucellosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, in particular to a primer set for detecting Brucella, a kit and a detection method. BACKGROUND

[0002] Brucellosis is a widespread and harmful zoonosis caused by Brucella. Brucella is a facultative intracellular parasitic gram-negative bacteria that can infect humans and more than 60 other mammals, seriously affecting human health and the development of animal husbandry. Therefore, it is important to develop a detection method with short time consumption, high sensitivity and high specificity for the diagnosis of Brucella infection. At present, the laboratory diagnosis methods of Brucellosis mainly include blood culture, Rose Bengal plate agglutination test (RBT), test tube agglutination test (SAT), anti-human globulin test (AGT), enzyme-linked immunosorbent assay (ELISA), immunogold technique (GICT), nucleic acid detection technology (NAT) and the like. Among them, blood culture takes 3-7 days, RBT is used for preliminary screening, and traditional serological tests such as SAT and AGT have false positive and false negative phenomena. The nucleic acid detection method has high sensitivity and good specificity, but requires complex operation steps, specific experimental instruments and professional operators.

[0003] At present, the laboratory diagnosis methods of Brucellosis mainly include blood culture, Rose Bengal plate agglutination test (RBT), test tube agglutination test (SAT), anti-human globulin test (AGT), enzyme-linked immunosorbent assay (ELISA), immunogold technique (GICT), nucleic acid detection technology (NAT) and the like. Among them, blood culture takes 3-7 days, RBT is used for preliminary screening, and traditional serological tests such as SAT and AGT have false positive and false negative phenomena. The nucleic acid detection method has high sensitivity and good specificity, but requires complex operation steps, specific experimental instruments and professional operators. Most of the above detection methods take a long time or have poor sensitivity, which is difficult to meet the needs of rapid diagnosis and accurate diagnosis. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a primer set for detecting Brucella, a kit and a detection method, which are applied to the clinical detection of suspected Brucella infection samples, not only shorten the time of conventional Brucella detection (blood culture), but also improve the sensitivity and specificity of traditional serological tests.

[0005] The technical problem to be solved by the present application is solved by the following technical scheme:

[0006] A primer set for detecting Brucella, the primer set is selected from any one or combination of (i) and (ii):

[0007] (i) primer set A1 for amplifying Brucella B1 target sequence (SEQ ID No. 1), comprising:

[0008] forward primer B1F1, the nucleotide sequence of which is shown as SEQ ID No. 3;

[0009] reverse primer B1R1, the nucleotide sequence of which is shown as SEQ ID No. 6;

[0010] (ii) primer set A2 for amplifying Brucella B2 target sequence (SEQ ID No. 2), comprising:

[0011] forward primer B2F1, the nucleotide sequence of which is shown as SEQ ID No. 8;

[0012] reverse primer B2R1, the nucleotide sequence of which is shown as SEQ ID No. 11.

[0013] Preferably, in the above technical solution, the primer set A1 further comprises crRNA1, the sequence of which is shown as SEQ ID No. 13; and the primer set A2 further comprises crRNA2, the sequence of which is shown as SEQ ID No. 14.

[0014] Use of a primer set in any one of the following: in detecting Brucella; in preparing a product for detecting Brucella; in a product for diagnosing Brucella infection; in a product for diagnosing brucellosis.

[0015] A kit for detecting Brucella, the kit comprising the primer set.

[0016] Preferably, in the above technical solution, the kit further comprises: a Cas12a protein and a single-stranded reporter molecule.

[0017] Preferably, in the above technical solution, the Cas12a protein is an LbCas12a protein; and the single-stranded reporter molecule is any one or both of: (i) 5'-FAM-TTATTATT-BHQ-3'; and (ii) 5'-FAM-TTATTATT-Biotin-3'.

[0018] A method for detecting Brucella for non-disease diagnosis purposes, comprising the following steps:

[0019] (1) extracting genomic DNA from a sample;

[0020] (2) using the A1 primer set and / or the A2 primer set of claim 1 to respectively perform RPA amplification on the genomic DNA extracted in step (1) as a template, to obtain amplification product 1 and / or amplification product 2.

[0021] (3): Mix the amplification product 1 and / or amplification product 2 obtained in step (2) with the CRISPR-Cas12a system for reaction, wherein the system contains Cas12a protein, crRNA corresponding to the selected primer set and single-stranded reporter molecule;

[0022] (4): The cleavage activity signal of the system is detected by side-flow chromatography test strips or fluorescence detection method to determine whether Brucella exists in the sample.

[0023] Preferably, in the above technical solution, in step (2), the RPA amplification conditions are 37 ℃ for 20 min; the CRISPR reaction conditions are 37 ℃ for 20-60 min.

[0024] Preferably, in the above technical solution, in step (3), the crRNA includes crRNA1 with a nucleotide sequence as shown in SEQ ID No. 13 targeting the B1 target sequence and / or crRNA2 with a nucleotide sequence as shown in SEQ ID No. 14 targeting the B2 target sequence.

[0025] Preferably, in the above technical solution, step (4) reads the result in any of the following ways:

[0026] Fluorescence method: Using 5'-FAM-TTATTATT-BHQ-3' as a single-chain reporter molecule, real-time readings are performed at 37 ℃ using the FAM channel of a fluorescence meter. The fluorescence signal in the FAM channel is monitored in real-time at 37 ℃ using the fluorescence meter. A significant increase in the fluorescence signal value is considered positive (generally, sterile ddH2O is used as a negative control; a positive result is defined as a real-time fluorescence value ≥ twice the fluorescence value of the negative control); or

[0027] LF method: Using 5'-FAM-TTATTATT-Biotin-3' as a single-chain reporter molecule, the sample is detected by lateral flow chromatography test strip. If both the T line and C line of the LF test strip are colored, or only the T line is colored, the sample is determined to contain Brucella. If the C line of the LF test strip is colored but the T line is not colored, the sample is determined to not contain Brucella.

[0028] The above-described technical solution of the present invention has the following beneficial effects:

[0029] The system provided by this invention is not only time-efficient, highly sensitive, and highly specific, but also can be implemented without relying on precision machinery. Both RPA reaction reagents and CRISPR-Cas12a reaction reagents can be commercially available kits. Detection can be completed by referring to the instructions. No specific experimental instruments or professional operators are required, enabling short-time, sensitive, specific, and visual detection. Attached Figure Description

[0030] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0031] Figure 1 For the screening of Brucella RPA primer pairs, a) is the screening of optimal primer pairs for systems constructed with B1-specific sequences. b) is the screening of optimal primer pairs for systems constructed with B2-specific sequences. c) is the screening of optimal primer concentrations and reaction times for RPA amplification systems.

[0032] Figure 2 Sensitivity evaluation of the RPA-CRISPR / Cas12a detection system. a) Sensitivity of the system constructed based on fluorescence signal intensity verification of the B1-specific sequence. b) Sensitivity of the system constructed based on fluorescence signal intensity verification of the B2-specific sequence. c) Sensitivity of the system constructed based on immunochromatographic test strip verification of the B1-specific sequence. d) Sensitivity of the system constructed based on immunochromatographic test strip verification of the B2-specific sequence.

[0033] Figure 3 To verify the specificity of the RPA-CRISPR / Cas12a detection system based on fluorescence intensity detection. a–c show the detection results of Brucella abortus RB51 with Gram-negative bacilli (a), Gram-positive cocci (b), and fungi (c) using the system constructed with the B1 specific sequence. d–f show the detection results of the system constructed with the B2 specific sequence.

[0034] Figure 4 To verify the specificity of the RPA-CRISPR / Cas12a detection system based on test strips. ab shows the detection system constructed using the B1 specific sequence, used to detect Brucella abortus RB51 against other bacteria (a) and fungi (b), respectively. c–d show the detection results using the detection system constructed using the B2 specific sequence.

[0035] Figure 5 The RPA-CRISPR / Cas12a system was initially validated in simulated clinical samples. a–b show systems constructed using the B2-specific sequence based on fluorescence intensity signal detection, used to detect different concentrations of Brucella abortus RB51 in serum samples (a) and whole blood samples (b). c–d show systems constructed using the B3-specific sequence, used to detect serum samples (c) and whole blood samples (d) at the same concentration range. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Unless otherwise specified, the equipment used in the experiments is well known to those skilled in the art.

[0038] This invention provides a method for visually detecting Brucella based on the RPA-CRISPR-Cas12a system, comprising: recombinase, RPA primers, polymerase, single-stranded binding protein, CrRNA, single-stranded reporter molecule (5'-FAM-TTATTATT-Biotin-3'), and LF test strip; the RPA primers include F and R; the nucleotide sequences of F and R are shown in Table 2, SEQ ID No. 3 to SEQ ID No. 12; the nucleotide sequence of CrRNA is shown in Table 2, SEQ ID No. 13 to SEQ ID No. 14; the nucleotide sequence of the single-stranded reporter molecule is 5'-TTATTATT-3'; a fluorescent labeling group is attached to the T base at the 5' end of the single-stranded reporter molecule, and biotin is attached to the T base at the 3' end of the single-stranded reporter molecule. During the reaction, the recombinase binds to the RPA primer to form a protein-DNA mixture, searching for homologous sequences on the Brucella DNA template. The polymerase initiates DNA synthesis, and the resulting amplification product contains a target sequence that can be recognized by CrRNA. CrRNA and Cas12a protein bind to form a complex. CrRNA guides Cas12a protein to recognize the target sequence and cleave it, subsequently cleaving a single-stranded reporter molecule. Since a fluorescent labeling group is attached to the T base at the 5' end of the single-stranded reporter molecule, the LF test strip is used to detect whether the single-stranded reporter molecule has been cleaved, thereby achieving visual detection of Brucella. The method for visual detection of Brucella based on the RPA-CRISPR-Cas12a system provided by this invention has the characteristics of short processing time, high sensitivity, and good specificity.

[0039] This invention provides a method for visually detecting Brucella based on the RPA-CRISPR-Cas12a system, comprising: recombinase, RPA primers, polymerase, single-stranded binding protein, CrRNA, single-stranded reporter molecule, and LF test strip; the RPA primers include A1 primer set sequences F and R and A2 primer set sequences F and R; the nucleotide sequence of A1 primer set F is shown in Table 2, SEQ ID No. 3, specifically B1F1: 5'-AACGATGATGCAAAAGCCAT-3'; the nucleotide sequence of A1 primer set R is shown in Table 2, SEQ ID No. 6, specifically B1R1: 5'-AGCTTGAATATTCCTGCATGGT-3'; the nucleotide sequence of A2 primer set F is shown in Table 2, SEQ ID No. 8, specifically B2F1: 5'-TCAAGTATGAGGCTGAAAAGCA-3'; the nucleotide sequence of A2 primer set R is shown in Table 2, SEQ ID No. 8, specifically B2F1: 5'-TCAAGTATGAGGCTGAAAAGCA-3'; As shown in No. 11, specifically B2R1: 5'-GGAATTTCATCGACACTCATCA-3'; the nucleotide sequence of the A1 primer set CrRNA is shown in Table 2, SEQ ID No. 13, specifically CrRNA-B1: uaauuucuacuaaguguagauUCCGUUCUCUCGGAGGAUAACCA; the nucleotide sequence of the A2 primer set CrRNA is shown in Table 2, SEQ ID No. 14, specifically CrRNA-B2: uaauuucuacuaaguguagauAUAAACACCGACAAGGCUCUGCA; the nucleotide sequence of the single-stranded reporter molecule is 5'-TTATTATT-3'; a fluorescent labeling group is attached to the T base at the 5' end of the single-stranded reporter molecule, and biotin is attached to the T base at the 3' end of the single-stranded reporter molecule.

[0040] This invention also provides a method for detecting Brucella based on the RPA-CRISPR-Cas12a system, comprising: recombinase, RPA primers, polymerase, single-stranded binding protein, CrRNA, single-stranded reporter molecule (5'-FAM-TTATTATT-BHQ-3'), and a fluorescence detection device. This method uses the same reagents and reaction system as the aforementioned LF test strip detection system, except that the single-stranded reporter molecule (probe) DNA-FAM-BIO is replaced with DNA-FAM-BHQ. Sterile ddH2O is used as a negative control. The detection process is performed in the FAM channel of a qPCR instrument at a reaction temperature of 37°C. Fluorescence values ​​are read once per minute for a total of 60 reads (60 minutes total) to observe changes in fluorescence signal intensity. A positive result is defined as a significant increase in fluorescence signal value (generally, a positive result is defined as a real-time fluorescence value ≥ twice the fluorescence value of the negative control).

[0041] The two detection systems provided by this invention are not only time-efficient, highly sensitive, and highly specific, but can also be implemented without relying on precision machinery. Both RPA and CRISPR-Cas12a reaction reagents can be commercially available kits, and the detection can be completed by referring to the instructions. No specific experimental instruments or professional operators are required, and short-time, sensitive, specific, and visual detection can be achieved.

[0042] In developing the detection system of this invention, multiple pairs of candidate RPA primers (see Table 2) and corresponding crRNA sequences were designed targeting Brucella B1 and B2 target sequences (SEQ ID No. 1 and SEQ ID No. 2). Through extensive and rigorous experimental screening and performance verification (such as sensitivity, specificity, and amplification efficiency), the primer pairs consisting of B1F1 (SEQ ID No. 3), B1R1 (SEQ ID No. 6) and B2F1 (SEQ ID No. 8), B2R1 (SEQ ID No. 11), along with their corresponding crRNA1 (SEQ ID No. 13) and crRNA2 (SEQ ID No. 14), were ultimately determined as the optimal combination.

[0043] Table 1 Brucella-specific target sequences

[0044]

[0045] Table 2. RPA amplification primer pairs designed for Brucella-specific target sequences.

[0046]

[0047] Note: The uppercase letters in crRNA are spacer sequences (guide sequences), which are complementary to the RPA amplification product sequence, while the lowercase letters are repeat sequences (anchor sequences), which are sequences that bind to the Cas12a protein.

[0048] Example 1: Visual Detection System Based on RPA-CRISPR-Cas12a System

[0049] A system for visually detecting Brucella based on the RPA-CRISPR-Cas12a system is disclosed. The system comprises RPA primers, CrRNA, a single-stranded reporter molecule, an LF test strip (EZassay's Lateral flow paper strip (for CRISPR SHERLOCK), catalog number HD-FMBO-96), an RPA kit (TwistDx's TwistAmp® Basic Kit, catalog number TABAS03KIT), and a Cas12a kit (LbaCas12a (Cpf1) protein enhancement version (from Lachnospiraceae), catalog number CAS-12E-010) purchased from Shenzhen Yizhi Biotechnology Co., Ltd.). The RPA primers include A1 primer set sequences F and R and A2 primer set sequences F and R.

[0050] The nucleotide sequence of primer group F of A1 is shown in Table 2, SEQ ID No. 3, specifically B1F1 5'AACGATGATGCAAAAGCCAT- 3';

[0051] The nucleotide sequence of primer group R of A1 is shown in Table 2, SEQ ID No. 6, specifically B1R1 5'AGCTTGAATATTCCTGCATGGT- 3';

[0052] The nucleotide sequence of primer group F of A2 is shown in Table 2, SEQ ID No. 8, specifically B2F1 5'TCAAGTATGAGGCTGAAAAGCA- 3';

[0053] The nucleotide sequence of primer group R in A2 is shown in Table 2, SEQ ID No. 11, specifically B2R1 5'GGAATTTCATCGACACTCATCA- 3';

[0054] The nucleotide sequence of CrRNA in primer group A1 is shown in Table 2, SEQ ID No. 13, specifically CrRNA-B1 uaauuucuacuaaguguagauUCCGUUCUCUCGGAGGAUAACCA;

[0055] The nucleotide sequence of CrRNA in primer group A2 is shown in Table 2, SEQ ID No. 14, specifically CrRNA-B2 uaauuucuacuaaguguagauAUAAACACCGACAAGGCUCUGCA;

[0056] The nucleotide sequence of the single-stranded reporter molecule is 5'-FAM-TTATTATT-Biotin-3'.

[0057] Example 2: Brucella detection method for non-disease diagnosis purposes

[0058] A method for detecting Brucella for non-diagnostic purposes comprises the following steps:

[0059] Using DNA from the suspected sample as a template, an RPA reaction was performed using the RPA primers and RPA kit in the system described in Example 1 to obtain the RPA amplification product.

[0060] The RPA reaction system, consisting of 50 μL of F, R, primer-free reconstitution buffer (29.5 μL), DNA template (1 μL), ddH2O (13.0 μL), and magnesium acetate (2.5 L), comprises: 2.0 μL of F, 2.0 μL of R, 29.5 μL of primer-free reconstitution buffer, 1 μL of DNA template, 13.0 μL of ddH2O, and 2.5 L of magnesium acetate. The concentrations of F and R in the reaction system are both 0.40 μM. The concentration of magnesium acetate is 40 mmol / L. The RPA reaction is carried out at 37°C for 20 min.

[0061] The RPA amplification product, CrRNA from the system described in Example 1, single-stranded reporter molecule and Cas12a kit were subjected to CRISPR-Cas12a reaction to obtain the reaction product;

[0062] The CRISPR-Cas12a reaction system, totaling 20 μL, consisted of: 2 μL of 10× Cas12a reaction buffer, 0.24 μL of 10 μM single-stranded reporter molecule (5'-FAM-TTATTATT-Biotin-3'), 1 μL of 1 μM crRNA, 1 μL of 1 μM LbaCas12a protein, 5 μL of RPA amplification product, and 10.76 μL of RNase-free deionized water. The CRISPR-Cas12a reaction was performed at 37°C for 60 min.

[0063] The reaction product was detected using the LF test strip in the system described in Example 1. The test was performed according to the instructions, and the presence of Brucella in the suspected sample was determined.

[0064] (1) If both the T line and the C line of the LF test strip are colored, or only the T line is colored, the sample is suspected to contain Brucella.

[0065] (2) If the C line of the LF test strip is colored but the T line is not colored, the sample to be tested is suspected to not contain Brucella.

[0066] (3) If neither the C line nor the T line of the LF test strip shows color, it means that the LF test strip is contaminated and a new test strip needs to be used for retesting.

[0067] Example 3 Sensitivity of the RPA-CRISPR / Cas12a method for fluorescence detection

[0068] The standard plasmid Plasmid-melitensis(n) was used for 10-fold serial dilutions, with concentrations ranging from 10... 5 From 1 copy / μL to 1 copy / μL, plasmids of various concentration gradients were used as templates for RPA amplification. The amplification products were then used for subsequent CRISPR / Cas12a fluorescence detection to evaluate the detection sensitivity of this method.

[0069] Depend on Figure 2 As shown in ab, the LOD detected by the fluorescence method of the detection system constructed with specific sequences B1 and B2 is 1 copy / μL.

[0070] Example 4: Specificity of the RPA-CRISPR / Cas12a method for fluorescence detection

[0071] To verify the specificity of the detection system, 12 standard bacterial strains that can cause bloodstream infections were selected, including: *Enterococcus casseliflavus*, *Enterococcus faecium*, *Staphylococcus epidermidis*, *Streptococcus pyogenes A*, *Enterococcus faecalis*, *Staphylococcus aureus*, *Shigellasonnei*, *Haemophilus influenzae*, *Klebsiella pneumoniae*, *Escherichia coli*, *Streptococcus pneumoniae*, and *Pseudomonas aeruginosa*; and 4 standard fungal strains, including: *Candida glabrata*, *Candida albicans*, *Candida guilliermondii*, and *Candida krusei*. The *Brucella abortus* RB51 vaccine strain was used as a positive control.

[0072] After purifying and culturing each strain, sufficient single colonies were picked and added to 1.5 mL centrifuge tubes containing 200 μL of sterile ddH2O. The tubes were heated in a 100℃ metal bath for 10 minutes, followed by centrifugation at 14,000 rpm for 10 minutes at 4℃. The supernatant was collected for later use. Sterile ddH2O was used as a negative control. The above-mentioned bacterial, fungal, and positive vaccine strains were tested. The detection process was performed in the FAM channel of a qPCR instrument at a reaction temperature of 37℃. Fluorescence values ​​were read once per minute for a total of 60 reads (total time 60 minutes) to observe changes in fluorescence signal intensity and thus evaluate the species specificity of the method.

[0073] Depend on Figure 3 It can be seen that in the fluorescence detection system, even when the nucleic acid concentration of non-specific bacteria is much higher than that of the target strain, only the fluorescence signal curve of the positive control group shows a significant increase, while the other curves remain horizontal. This indicates that the system is only suitable for detecting Brucella and has high specificity.

[0074] Example 5: Sensitivity of the RPA-CRISPR / Cas12a detection method for LF test strips

[0075] To visualize the test results, we combined immunochromatography with a newly developed RPA-CRISPR / Cas12a detection method to construct a rapid on-site detection system based on Lateral Flow Strip (LFS). This method uses the same reagents and reaction system as the fluorescence detection system, except that the fluorescent reporter probe DNA-FAM-BHQ is replaced with DNA-FAM-BIO.

[0076] A 20 μL reaction mixture containing 2 μL 10×Cas12a reaction buffer, 0.24 μL DNA-FAM-BIO (10 μM) reporter probe, 1 μL crRNA (1 μM), 1 μL LbaCas12a protein (1 μM), 5 μL RPA amplification product, and 10.76 μL RNase-free water was mixed and incubated at 37°C for 45 minutes. Then, 10 μL of the product was diluted 1:3. A lateral chromatography strip was inserted into the reaction product, and the results were read within 10 minutes. The strip has two bands: a control line at the bottom and a test line at the top. If the test line is red, the result is positive, indicating that the nucleic acid probe has been specifically cleaved by the Cas12a enzyme, and the enzyme activity has been activated. If the test line is not colored, and only the control line is colored, the result is negative.

[0077] Depend on Figure 2According to the CD, the LOD of the detection system constructed with specific sequence B1 is 10 copies / μL, and the LOD of the detection system constructed with sequence B2 is 1 copy / μL.

[0078] Example 6: Specificity of the RPA-CRISPR / Cas12a detection method for LF test strips

[0079] To verify the specificity of this detection system, we selected 12 standard bacterial strains that can cause bloodstream infections, including: *Enterococcus casseliflavus*, *Enterococcus faecium*, *Staphylococcus epidermidis*, *Streptococcus pyogenes A*, *Enterococcus faecalis*, *Staphylococcus aureus*, *Shigella sonnei*, *Haemophilus influenzae*, *Klebsiella pneumoniae*, *Escherichia coli*, *Streptococcus pneumoniae*, and *Pseudomonas aeruginosa*; and 4 standard fungal strains, including: *Candida glabrata*, *Candida albicans*, *Candida guilliermondii*, and *Candidakrusei*. The *Brucella abortus* RB51 vaccine strain was used as a positive control.

[0080] After culturing each strain separately, a sufficient number of single colonies were picked and added to a 1.5 mL centrifuge tube containing 200 μL of sterile ddH2O. The tube was heated in a 100°C metal bath for 10 minutes, followed by centrifugation at 14,000 rpm for 10 minutes at 4°C. The supernatant was collected for later use. Sterile ddH2O was used as a negative control, and nucleic acids from the above-mentioned bacteria, fungi, and positive vaccine strains were used for detection.

[0081] Mix the following 20 μL reaction mixture (containing 2 μL 10×Cas12a reaction buffer, 0.24 μL DNA-FAM-BIO (10 μM) reporter probe, 1 μL crRNA (1 μM), 1 μL LbaCas12a protein (1 μM), 5 μL RPA amplification product, and 10.76 μL RNase-free water) and incubate at 37°C for 45 minutes. Then, take 10 μL of the product and dilute it 1:3. Insert the lateral chromatography strip into the reaction product and read the results within 10 minutes. The strip has two bands: a control line at the bottom and a test line at the top. If the test line is red, the result is positive, indicating that the nucleic acid probe has been specifically cleaved by the Cas12a enzyme and the enzyme activity has been activated. If the test line is not colored, and only the control line is colored, the result is negative.

[0082] Depend on Figure 4 It can be seen that by using test strips for testing and visualizing the results, this detection system can only detect positive strains.

[0083] Example 7: Simulated Clinical Sample Testing

[0084] Clinical simulants containing different concentrations of Brucella vaccine strain RB51 were prepared using serum and whole blood, respectively, ranging from 4.65 × 10³ CFU / mL to 0.465 CFU / mL.

[0085] Depend on Figure 5 It was found that two detection systems, constructed using the RPA-CRISPR / Cas12a method and based on specific sequences B1 and B2, were used for detection via fluorescence detection and test strip methods, respectively. Both systems achieved detection values ​​as low as 4.65 × 10⁻⁶ in serum and whole blood using the fluorescence detection method. 2 The detection limit for Brucella is CFU / mL; while the detection limit for the test strip method is 4.65 × 10³ CFU / mL.

[0086] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. Use of a primer set for detecting Brucella in any one of the following: for detecting Brucella for non-disease diagnosis purposes; for preparing a product for detecting Brucella, the primer set is selected from (i) or (ii) below: (i) primer set A1 for amplifying Brucella B1 target sequence (SEQ ID No. 1), comprising: forward primer B1F1, the nucleotide sequence of which is shown as SEQ ID No. 3; reverse primer B1R1, the nucleotide sequence of which is shown as SEQ ID No. 6; (ii) primer set A2 for amplifying Brucella B2 target sequence (SEQ ID No. 2), comprising: forward primer B2F1, the nucleotide sequence of which is shown as SEQ ID No. 8; reverse primer B2R1, the nucleotide sequence of which is shown as SEQ ID No. 11; the primer set A1 further comprises crRNA1, the sequence of which is shown as SEQ ID No. 13; and the primer set A2 further comprises crRNA2, the sequence of which is shown as SEQ ID No.

14.

2. A method for detecting Brucella for a purpose other than disease diagnosis, characterized by, comprising the following steps: (1) extracting genomic DNA from a sample; (2) using the genomic DNA extracted in step (1) as a template, performing RPA amplification with the A1 primer set or the A2 primer set according to claim 1 to obtain amplification product 1 or amplification product 2, respectively; (3) mixing the amplification product 1 or the amplification product 2 obtained in step (2) with a CRISPR-Cas12a system to perform a reaction, wherein the system contains Cas12a protein, crRNA corresponding to the selected primer set, and a single-stranded reporter molecule; (4) detecting the cleavage activity signal of the system by lateral flow chromatographic test strip or fluorescence detection method to determine whether Brucella is present in the sample.

3. The detection method according to claim 2, characterized in that, In step (2), the RPA amplification conditions are 37°C for 20 min; and the CRISPR reaction conditions are 37°C for 20-60 min.

4. The detection method according to claim 2, characterized in that, In step (3), the Cas12a protein is LbCas12a protein; and the single-stranded reporter molecule is any one or both of (i) 5'-FAM-TTATTATT-BHQ-3' and (ii) 5'-FAM-TTATTATT-Biotin-3'.

5. The detection method according to claim 2, characterized in that, In step (4), the result is read by any one of the following methods: fluorescence method: using 5'-FAM-TTATTATT-BHQ-3' as a single-stranded reporter molecule, reading in real time at 37°C under the FAM channel of a fluorescence reader, and monitoring the fluorescence signal of the FAM channel in real time at 37°C in the fluorescence reader, and when the fluorescence signal value shows a significant increase, it is determined to be positive; or LF method: using 5'-FAM-TTATTATT-Biotin-3' as a single-stranded reporter molecule, detecting by lateral flow chromatographic test strip, and if both the T line and the C line of the LF test strip develop color, or only the T line develops color, it is determined that the sample contains Brucella; and if the C line of the LF test strip develops color and the T line does not develop color, it is determined that the sample does not contain Brucella.

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