A primer-probe combination and detection method for rapid detection of brucellosis pathogen

Abstract

This invention discloses a primer-probe combination and detection method for rapid detection of Brucella pathogens, including a first primer-probe set targeting Brucella IS711, a second primer-probe set targeting Brucella BMEI1827, and an internal standard primer-probe set for a synthetic internal standard template. In the first primer-probe set, the nucleotides at positions 5-9 and 15-19 are modified with locked nucleic acids; in the second primer-probe set, the nucleotides at positions 8-12 are modified with locked nucleic acids. The probes of the internal standard primer-probe set are labeled with a VIC reporter group at the 5' end and a BHQ2 quencher group at the 3' end. The advantages of this invention are: improved detection specificity and sensitivity, effective avoidance of missed and false detections, and the ability to monitor the effectiveness of the detection system throughout the process by introducing a synthetic internal standard, thus solving the false negative problem; combined with the UNG enzyme-dUTP anti-contamination system and closed-tube detection mode, it eliminates false positive results caused by aerosol contamination.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a primer-probe combination and detection method for rapid detection of Brucella pathogens. Background Technology

[0002] Brucellosis is a serious zoonotic infectious disease that severely threatens livestock production and human health. It is widespread globally, damaging the reproductive systems of livestock such as cattle and sheep, leading to abortion, infertility, and other serious economic losses. Human infection typically occurs through contact with infected animals or their products. The clinical manifestations are complex, it easily becomes chronic, and it is difficult to cure completely.

[0003] Currently, the main methods for detecting the pathogens of brucellosis include bacterial isolation and culture, serological testing, and molecular biological detection. Bacterial isolation and culture is the "gold standard" for diagnosis, but it is complex to perform, has high biosafety requirements, and a long culture period, making it unsuitable for rapid diagnosis. Serological testing is relatively simple to perform, but it suffers from insufficient sensitivity and specificity, and cannot distinguish between current and past infections, easily leading to false positives or false negatives. While conventional polymerase chain reaction (PCR) technology improves the sensitivity and specificity of detection, it requires opening the gel electrophoresis container after amplification, which can easily generate aerosol contamination and cause false positives.

[0004] Therefore, in order to address the above-mentioned technical problems, it is necessary to provide a primer-probe combination and detection method for rapid detection of Brucella pathogens. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, the present invention aims to provide a primer-probe combination and detection method for rapid detection of Brucella pathogens, thereby solving the problems mentioned in the background art.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: This invention provides a primer and probe combination for rapid detection of Brucella pathogens, including a first primer and probe set targeting Brucella IS711, a second primer and probe set targeting Brucella BMEI1827, and an internal standard primer and probe set targeting a synthetic internal standard template.

[0007] In one or more embodiments of the present invention, the nucleotides at positions 5-9 and 15-19 of the probes in the first primer-probe set are modified with locked nucleic acids; the nucleotides at positions 8-12 of the probes in the second primer-probe set are modified with locked nucleic acids; the probes of the internal standard primer-probe set are labeled with a VIC reporter group at the 5' end and a BHQ2 quencher group at the 3' end.

[0008] A rapid detection method for brucellosis pathogens includes the following steps: S1. Obtain the sample to be tested, add an artificially synthesized internal standard template with a known copy number to the sample to be tested, and then perform nucleic acid co-extraction on the spiked sample to obtain a nucleic acid sample to be tested containing both sample nucleic acid and internal standard nucleic acid. S2. Mix the nucleic acid sample to be tested with the primer and probe combination and the UNG enzyme-dUTP real-time fluorescence quantitative PCR reaction solution, and centrifuge to obtain the amplification reaction system; The primer-probe combination includes a first primer-probe set targeting Brucella IS711, a second primer-probe set targeting Brucella BMEI1827, and an internal standard primer-probe set targeting the artificially synthesized internal standard template. The UNG enzyme-dUTP real-time fluorescence quantitative PCR reaction solution contains hot-start DNA polymerase, dUTP, dNTPs, UNG enzyme, magnesium ions, and reaction buffer. S3: Place the amplification reaction system in a multi-channel real-time PCR instrument, run the preset rapid amplification program, collect fluorescence signals in real time in the FAM channel, VIC channel and Cy5 channel, and generate the first amplification curve data corresponding to the IS711 target, the second amplification curve data corresponding to the BMEI1827 target and the internal standard amplification curve data corresponding to the internal standard, respectively. S4: Obtain the internal standard amplification curve data, determine whether there is an S-type amplification curve in the internal standard amplification curve data and calculate the internal standard cycling threshold; if there is an S-type amplification curve and the internal standard cycling threshold is within the preset effective range, then the detection is determined to be valid and proceed to step 5; if there is no S-type amplification curve or the internal standard cycling threshold is outside the preset effective range, then the detection is determined to be invalid and the detection is terminated. S5: After S4 determines that the detection is valid, acquire the first amplification curve data and the second amplification curve data, determine whether there is an S-type amplification curve in the first amplification curve data or the second amplification curve data, and calculate the corresponding target cycle threshold. If at least one target exhibits an S-type amplification curve and the cycle threshold of that target is less than a preset positive threshold, then the sample to be tested is determined to be positive for Brucella nucleic acid. If neither target exhibits an S-type amplification curve, or if the cycle thresholds of both targets are greater than or equal to the preset positive threshold, then the sample to be tested is determined to be Brucella nucleic acid negative.

[0009] In one or more embodiments of the present invention, the sample to be tested in step S1 is one of whole blood, serum, milk sample or tissue homogenate; the nucleic acid co-extraction process uses a magnetic bead extraction kit; the artificially synthesized internal standard template is a DNA fragment that does not have any homology with any known pathogen sequence, and the copy number of the template is 500-2000 copies per reaction.

[0010] In one or more embodiments of the present invention, in the UNG enzyme-dUTP real-time fluorescence quantitative PCR reaction solution in step S2, the concentration of the UNG enzyme is 0.01-0.1 U / μL, and the molar percentage of dUTP in the total dNTPs is 40%-60%; the total volume of the amplification reaction system is 25 μL, and the volume of the nucleic acid sample to be tested is 5 μL.

[0011] In one or more embodiments of the present invention, the preset rapid amplification program in step S3 includes: UNG enzyme treatment at 50°C for 2 minutes; a hot start at 95°C for 3 minutes; and 45 cycles of amplification, each cycle including denaturation at 95°C for 5 seconds and annealing extension at 60°C for 20 seconds, and simultaneously acquiring fluorescence signals from three channels, FAM, VIC and Cy5, at the end of the annealing extension step in each cycle.

[0012] In one or more embodiments of the present invention, the preset effective range in step S4 is that the internal standard cyclic threshold is between 25 and 35. In one or more embodiments of the present invention, the preset positive threshold in step S5 is 35.

[0013] The beneficial effects of this invention are as follows: 1. It can improve the specificity and sensitivity of detection, effectively avoid false negatives and false negatives, and solve the problem of false negatives by introducing artificially synthesized internal standards to monitor the effectiveness of the detection system throughout the process. 2. Combined with the UNG enzyme-dUTP anti-contamination system and closed-tube detection mode, it eliminates false positive results caused by aerosol contamination. At the same time, it adopts a rapid amplification program, and the entire detection time can be controlled within 40 minutes, which is much faster than traditional bacterial isolation and culture methods. It can meet the rapid screening needs of clinical, breeding farms and quarantine departments, and is compatible with a variety of clinical samples. It has wide applicability and high accuracy. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a gradient detection amplification curve of a fluorescent quantitative standard for a rapid detection method of brucellosis pathogen in one embodiment of the present invention; Figure 2 This is a diffusion curve of different concentrations of a rapid detection method for brucellosis pathogens according to an embodiment of the present invention; Figure 3 This is a target curve diagram of a rapid detection method for brucellosis pathogens according to an embodiment of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] One embodiment of the present invention provides a primer and probe combination for rapid detection of Brucella pathogens, comprising three sets of specific primer and probe systems: a first primer and probe set targeting the Brucella IS711 gene, a second primer and probe set targeting the Brucella BMEI1827 gene, and an internal control primer and probe set targeting a synthetic internal control template. The dual-target synergistic detection significantly improves the specificity and sensitivity of Brucella detection. The internal control system monitors the entire process of nucleic acid extraction and amplification, avoiding false negatives and invalid detection results.

[0018] The first primer and probe set is designed for the conserved and specific IS711 target of Brucella. The probe employs locked nucleic acid modification technology, specifically modifying nucleotides 5-9 and 15-19. Locked nucleic acid modification significantly enhances the binding affinity between the probe and the target nucleic acid, improves probe annealing specificity, reduces non-specific binding, and greatly increases the detection rate of low-copy pathogen samples. Simultaneously, the probe is labeled with a FAM reporter group at its 5' end and a BHQ1 quencher group at its 3' end, corresponding to the FAM detection channel of a quantitative real-time PCR instrument, specifically recognizing the IS711 target amplification signal.

[0019] The second primer and probe set is designed specifically for the Brucella adenosalis target BMEI1827. The probes are also optimized using locked nucleic acid modification, with the modification site being nucleotides 8-12. This site-specific locked nucleic acid modification enhances probe stability and targeting binding ability, effectively distinguishing Brucella from other homologous pathogens and avoiding cross-reactivity. This probe is matched with the Cy5 fluorescence detection channel, forming a multi-channel differential fluorescence detection system with the first primer and probe set and the internal control probe, enabling single-tube multiplex amplification detection.

[0020] The internal standard primer-probe set is designed for artificially synthesized internal standard templates. These templates are specific, artificially synthesized DNA fragments with no homology to any known pathogen sequences, and will not cause non-specific amplification with Brucella nucleic acid or nucleic acids of common clinical pathogens, thus not interfering with target detection results. The internal standard probe is labeled with a VIC reporter group at its 5' end and a BHQ2 quencher group at its 3' end, corresponding to the VIC detection channel. This allows for continuous monitoring of sample nucleic acid extraction efficiency, PCR amplification system effectiveness, and instrument operating status, accurately determining the validity of the detection results.

[0021] like Figures 1 to 3 As shown in one embodiment of the present invention, a rapid detection method for brucellosis pathogens uses UNG enzyme-dUTP real-time fluorescence quantitative PCR reaction solution as the core reaction system. A 25 μL standardized amplification system is constructed by combining the above-mentioned primer and probe combination. All reagents are nuclease-free and pyrogen-free to avoid exogenous nucleic acid contamination and enzyme activity failure. The specific reagent components and concentration ratios strictly follow the optimal parameter standards.

[0022] The core components of the UNG enzyme-dUTP real-time quantitative PCR reaction solution include hot-start DNA polymerase, dUTP, conventional dNTPs, UNG enzyme, magnesium ions, and a dedicated reaction buffer. The UNG enzyme concentration is strictly controlled at 0.01-0.1 U / μL, preferably 0.05 U / μL. This concentration efficiently degrades residual dU fragments in the PCR amplification products, completely eliminating cross-contamination in the laboratory caused by aerosols, while not damaging the native template nucleic acid and not affecting normal amplification efficiency. The molar percentage of dUTP in the total dNTPs is controlled at 40%-60%, preferably 50%. Through a reasonable ratio of dUTP to dTTP, both anti-contamination effect and amplification specificity and efficiency are balanced.

[0023] The hot-start DNA polymerase uses a high-temperature resistant and highly specific polymerase, which can avoid the amplification of non-specific primer dimers at room temperature and improve detection accuracy. The magnesium ion concentration is 2.0-3.0 mmol / L, preferably 2.5 mmol / L, to provide the optimal ionic environment for polymerase amplification and primer-probe binding. The reaction buffer is a 10× dedicated PCR buffer containing Tris-HCl, potassium chloride and other components to maintain the acid-base balance and ionic stability of the system.

[0024] The standardized 25 μL amplification reaction system was prepared with the following components: 5 μL of nucleic acid sample to be tested; the final concentrations of the upstream and downstream primers (first primer / probe set, second primer / probe set, and internal standard primer / probe set) were all 0.2 μmol / L; the final concentration of each probe was 0.1 μmol / L; 12.5 μL of UNG enzyme-dUTP PCR reaction solution was added; and the remaining volume was brought to 25 μL with enzyme-free, sterile ultrapure water. The system ratios are precise and controllable, compatible with various multi-channel real-time quantitative PCR instruments, and exhibit excellent repeatability and stability.

[0025] The specific testing procedures are as follows: Obtain the samples to be tested, and precisely add a synthetic internal standard template to each sample. The amount added is controlled at 500-2000 copies per reaction, preferably 1000 copies. This copy number ensures that the Ct value of the internal standard amplification remains stable within the effective range of 25-35, allowing for precise monitoring of the entire extraction and amplification process without interfering with the amplification and detection of Brucella target nucleic acid. After adding the internal standard template, thoroughly vortex to ensure complete integration of the template with the sample matrix. Subsequently, a commercial magnetic bead nucleic acid extraction kit is used for co-extraction of nucleic acids, simultaneously extracting Brucella nucleic acid and the synthetic internal standard nucleic acid from the sample. Finally, 50 μL of the nucleic acid sample to be tested is obtained by elution. Negative and positive controls are extracted simultaneously throughout the process to eliminate contamination and operational errors during extraction.

[0026] The magnetic bead extraction procedure strictly follows the kit instructions, involving five steps: lysis, binding, washing, drying, and elution. This process thoroughly removes impurities such as proteins, lipids, and pigments from the sample, ensuring the purity and integrity of the extracted nucleic acid. The nucleic acid purity OD260 / OD280 is controlled between 1.8 and 2.0, meeting the requirements for quantitative real-time PCR amplification.

[0027] PCR amplification systems were prepared in a clean operating bench for nucleic acid testing, strictly adhering to the principle of zoned operation. Sample processing, system preparation, and amplification detection were carried out in separate zones to prevent aerosol contamination. Following the standard 25μL system, UNG enzyme-dUTP real-time quantitative PCR reaction solution, three sets of primer and probe combinations, and enzyme-free ultrapure water were added sequentially. Finally, 5μL of the nucleic acid sample to be tested was added to avoid premature contact between the nucleic acid template and primers, which could lead to non-specific binding.

[0028] After the system was prepared, the PCR reaction tubes were briefly centrifuged in a mini centrifuge for 30 seconds at 3000 rpm to ensure that all residual liquid on the tube wall was centrifuged to the bottom of the tube, guaranteeing thorough mixing of the reaction system and eliminating any air bubbles that could affect the accuracy of fluorescence signal acquisition. Simultaneously, blank control (enzyme-free water replacing nucleic acid samples), negative control (nucleic acid extracted from healthy samples), and positive control (Brucella standard plasmid nucleic acid) were set up. Each group of samples was tested in triplicate to ensure the reproducibility of the test results.

[0029] The centrifuged PCR reaction tubes were placed into the sample slot of a multi-channel quantitative PCR instrument. Sample and channel information were accurately entered, and the simultaneous acquisition modes of the three channels (FAM, VIC, and Cy5) were set accordingly, corresponding to the fluorescence signals of the IS711 target, internal standard, and BMEI1827 target, respectively. The pre-set rapid amplification program of this invention was then run. The entire process is highly efficient and streamlined, with a total detection time of no more than 40 minutes, meeting the requirements for rapid detection.

[0030] The specific parameters of the preset rapid amplification program are as follows: The first step is UNG enzyme anti-contamination treatment, which involves incubation at 50℃ for 2 minutes to completely degrade any residual dU contaminants that may exist in the system; the second step is hot-start activation treatment, which involves incubation at 95℃ for 3 minutes to fully activate the hot-start DNA polymerase and simultaneously denature and denature the template nucleic acid; the third step is the cyclic amplification stage, which consists of a total of 45 amplification cycles. Each cycle includes denaturation at 95℃ for 5 seconds and annealing extension at 60℃ for 20 seconds. The amplification conditions have been optimized to significantly shorten the time of a single cycle and improve detection efficiency.

[0031] The fluorescence signal acquisition is set to simultaneously acquire fluorescence signals from three channels, FAM, VIC, and Cy5, after each cycle of annealing and extension. The instrument records the fluorescence intensity changes in real time, automatically generates the first amplification curve for the IS711 target, the second amplification curve for the BMEI1827 target, and the internal standard amplification curve, and stores the cycle threshold (Ct value) data in real time to provide data support for subsequent result determination.

[0032] After the amplification program is completed, the internal standard amplification curve data and Ct value are retrieved first to determine the validity of the detection. Only when the detection is valid can the target detection result be effective, thus completely avoiding false negative results caused by nucleic acid extraction failure, system failure, instrument failure, or interference from sample inhibitors.

[0033] The preset criteria for determining the validity of the internal standard in this invention are as follows: if the internal standard amplification curve exhibits a typical S-shaped amplification curve and the internal standard Ct value is within the range of 25-35, the test is deemed to be performed correctly, the system is effective, and the results are reliable, allowing the test to proceed to the subsequent target result determination process. If the internal standard does not exhibit an S-shaped amplification curve, or if the internal standard Ct value is <25 (inner standard over-amplification, system contamination) or >35 (low nucleic acid extraction efficiency, presence of inhibitors in the system, amplification failure), the test is directly deemed invalid, all sample data are discarded, and sample processing and amplification testing must be performed again.

[0034] Under the premise that the test results are deemed valid, the amplification curves of IS711 and BMEI1827 dual targets and the corresponding Ct values ​​are retrieved, and the sample results are judged according to the unified positive threshold standard. The preset positive threshold of this invention is 35.

[0035] Positive criteria: If at least one of the two targets shows a typical S-type specific amplification curve and the corresponding target Ct value is <35, the sample can be determined to be positive for Brucella nucleic acid, indicating the presence of Brucella pathogen in the sample. The detection results are accurate and reliable. Dual-target complementary detection can effectively avoid the problem of missed detection caused by single-target mutation.

[0036] Negative determination criteria: If neither of the two specific targets, IS711 and BMEI1827, shows an S-shaped amplification curve, or if the Ct values ​​of both targets are ≥35, the sample is determined to be negative for Brucella nucleic acid and free from Brucella pathogen infection.

[0037] To verify the specificity, sensitivity, repeatability, and anti-contamination ability of the detection method of the present invention, multiple control experiments were set up, and the system of the present invention was compared with the conventional single-target PCR detection system.

[0038] Specificity experiment: Nucleic acids of various subtypes of Brucella, Escherichia coli, Salmonella, Mycobacterium tuberculosis and other common human and animal pathogens were selected as test samples and detected using the dual-target system of this invention. The results showed that only Brucella samples showed specific amplification curves, while other pathogens did not show amplification signals. The specificity was 100%, with no cross-reaction. The nucleic acid-modified probe significantly improved the detection specificity.

[0039] Sensitivity test: Brucella standard plasmids were serially diluted to 10-10000 copies / reaction and detected using the system of this invention. Low concentrations of pathogens as low as 50 copies / reaction can be stably detected. Compared with traditional detection methods, the sensitivity is improved by 2-3 times, and accurate detection of trace infection samples can be achieved.

[0040] Repeatability test: High, medium and low concentration positive samples were selected, and 6 replicate wells were set in each group. Three batches were tested continuously. The coefficient of variation of Ct value of each target was <2%, which showed excellent repeatability and met the clinical and quarantine testing standards.

[0041] Contamination prevention verification: After treatment with the UNG enzyme-dUTP system, contaminated samples containing amplification products showed no false positive amplification, completely solving the aerosol contamination problem of traditional PCR technology and making it suitable for large-scale sample testing scenarios in laboratories. Simultaneously, the internal standard system can effectively identify problems such as nucleic acid extraction failure, sample inhibition, and incorrect system ratios, achieving a 100% invalid detection rejection rate and significantly improving detection accuracy.

[0042] Obviously, the above-described embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. A primer-probe combination for rapid detection of brucellosis pathogen, characterized in that, include: The first primer and probe set targeting Brucella IS711; A second primer and probe set targeting Brucella BMEI1827; An internal standard primer-probe set for an artificially synthesized internal standard template.

2. The primer-probe combination for rapid detection of brucellosis pathogen as described in claim 1, characterized in that, In the probes of the first primer-probe set, the nucleotides at positions 5-9 and 15-19 are modified with locked nucleic acids; In the probes of the second primer-probe set, the nucleotides at positions 8-12 are modified with locked nucleic acids; The probes of the internal standard primer-probe set are labeled with a VIC reporter group at the 5' end and a BHQ2 quencher group at the 3' end.

3. A rapid detection method for brucellosis pathogens, using the primer-probe combination as described in claim 1, characterized in that, Includes the following steps: S1. Obtain the sample to be tested, add an artificially synthesized internal standard template with a known copy number to the sample to be tested, and then perform nucleic acid co-extraction on the spiked sample to obtain a nucleic acid sample to be tested containing both sample nucleic acid and internal standard nucleic acid. S2. Mix the nucleic acid sample to be tested with the primer and probe combination and the UNG enzyme-dUTP real-time fluorescence quantitative PCR reaction solution, and centrifuge to obtain the amplification reaction system; The primer-probe combination includes a first primer-probe set targeting Brucella IS711, a second primer-probe set targeting Brucella BMEI1827, and an internal standard primer-probe set targeting the artificially synthesized internal standard template. The UNG enzyme-dUTP real-time fluorescence quantitative PCR reaction solution contains hot-start DNA polymerase, dUTP, dNTPs, UNG enzyme, magnesium ions, and reaction buffer. S3: Place the amplification reaction system in a multi-channel real-time PCR instrument, run the preset rapid amplification program, collect fluorescence signals in real time in the FAM channel, VIC channel and Cy5 channel, and generate the first amplification curve data corresponding to the IS711 target, the second amplification curve data corresponding to the BMEI1827 target and the internal standard amplification curve data corresponding to the internal standard, respectively. S4: Obtain the internal standard amplification curve data, determine whether there is an S-type amplification curve in the internal standard amplification curve data and calculate the internal standard cycling threshold; if there is an S-type amplification curve and the internal standard cycling threshold is within the preset effective range, then the detection is determined to be valid and proceed to step 5; if there is no S-type amplification curve or the internal standard cycling threshold is outside the preset effective range, then the detection is determined to be invalid and the detection is terminated. S5: After S4 determines that the detection is valid, acquire the first amplification curve data and the second amplification curve data, determine whether there is an S-type amplification curve in the first amplification curve data or the second amplification curve data, and calculate the corresponding target cycle threshold. If at least one target exhibits an S-type amplification curve and the cycle threshold of that target is less than a preset positive threshold, then the sample to be tested is determined to be positive for Brucella nucleic acid. If neither target exhibits an S-type amplification curve, or if the cycle thresholds of both targets are greater than or equal to the preset positive threshold, then the sample to be tested is determined to be Brucella nucleic acid negative.

4. The rapid detection method for brucellosis pathogen as described in claim 3, characterized in that, The sample to be tested in step S1 is one of whole blood, serum, milk sample or tissue homogenate; the nucleic acid co-extraction process uses a magnetic bead extraction kit; the artificially synthesized internal standard template is a DNA fragment that does not have any homology with any known pathogen sequence, and its copy number is added at 500-2000 copies per reaction.

5. The rapid detection method for brucellosis pathogen as described in claim 3, characterized in that, In step S2, the concentration of the UNG enzyme in the UNG enzyme-dUTP real-time fluorescence quantitative PCR reaction solution is 0.01-0.1 U / μL, and the molar percentage of dUTP in the total dNTPs is 40%-60%; the total volume of the amplification reaction system is 25 μL, and the volume of the nucleic acid sample to be tested is 5 μL.

6. The rapid detection method for brucellosis pathogen as described in claim 3, characterized in that, The preset rapid amplification program in step S3 includes: UNG enzyme treatment at 50°C for 2 minutes; hot start at 95°C for 3 minutes; and 45 cycles of amplification, each cycle including denaturation at 95°C for 5 seconds and annealing extension at 60°C for 20 seconds, and simultaneously acquiring fluorescence signals from the three channels FAM, VIC and Cy5 at the end of the annealing extension step in each cycle.

7. The rapid detection method for brucellosis pathogen as described in claim 3, characterized in that, The preset effective range in step S4 is that the internal standard cyclic threshold is between 25 and 35.

8. The rapid detection method for brucellosis pathogen as described in claim 3, characterized in that, The preset positive threshold in step S5 is 35.

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