A primer set, kit and use for differentiating BA0711 vaccine strain

Abstract

The application belongs to the field of biology, and discloses a primer set for distinguishing BA0711 vaccine strains, which comprises a first primer pair taking a Brucella BCSP31 gene as a target gene and a second primer pair taking a Brucella cspA gene as a target gene. The application takes the Brucella BCSP31 gene and the Brucella cspA gene as the target genes, can judge whether an animal is infected with Brucella by screening the Brucella BCSP31 gene, and can judge whether the animal is inoculated with the BA0711 vaccine strain by screening the Brucella cspA gene. Meanwhile, the application also provides a kit based on the primer set and uses.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a primer set, reagent kit, and uses for distinguishing BA0711 vaccine strains. Background Technology

[0002] In 2023, the Ministry of Agriculture and Rural Affairs approved Brucella live vaccine (BA0711 strain) as a new veterinary drug. BA0711 strain was developed from Brucella melitensis biotype 3 and has the characteristics of being suitable for pregnant animals and cattle and sheep.

[0003] Patent application CN119639926A, entitled "PCR Kit, Method, and Application for Identifying Brucella Pathogens," discloses a PCR kit, method, and application for identifying Brucella pathogens, including primer pairs and probes for simultaneously detecting Brucella BA0711 vaccine strain, M5-90△26 vaccine strain, Rev.1 vaccine strain, and wild-type strain; wherein the site for identifying Brucella BA0711 vaccine strain is a unique deletion sequence of Brucella BA0711 vaccine strain. There is limited research in this field on the Brucella BA0711 vaccine strain.

[0004] The technical problem to be solved in this case is: how to identify the BA0711 strain based on the new site. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a primer set for distinguishing the BA0711 vaccine strain. The present invention uses the Brucella BCSP31 gene and the Brucella cspA gene as target genes. By screening the Brucella BCSP31 gene, it can be determined whether an animal is infected with Brucella, and by screening the Brucella cspA gene, it can be determined whether an animal has been vaccinated with the BA0711 vaccine strain.

[0006] In addition, the present invention also provides a kit and its uses based on this primer set.

[0007] To achieve this objective, the present invention adopts the following technical solution: A primer set for distinguishing BA0711 vaccine strains includes a first primer pair targeting Brucella BCSP31 gene and a second primer pair targeting Brucella cspA gene. The first primer pair includes a first upstream primer and a first downstream primer; The second primer pair includes a second upstream primer and a second downstream primer, or a third upstream primer and a third downstream primer; The nucleotide sequence of the first upstream primer is shown in SEQ ID NO.1; The nucleotide sequence of the first downstream primer is shown in SEQ ID NO.2; The nucleotide sequence of the second upstream primer is shown in SEQ ID NO.3; The nucleotide sequence of the second downstream primer is shown in SEQ ID NO.4; The nucleotide sequence of the third upstream primer is shown in SEQ ID NO.5; The nucleotide sequence of the third downstream primer is shown in SEQ ID NO.6.

[0008] In addition, the present invention also discloses the use of a kit prepared using the primer set described above; the kit is used to distinguish between the BA0711 vaccine strain and the Brucella wild-type strain.

[0009] Finally, the present invention also discloses a reagent kit, comprising a first reagent kit and a second reagent kit; The first kit includes a first primer pair; the second kit includes a second primer pair. The first primer pair includes a first upstream primer and a first downstream primer; The second primer pair includes a second upstream primer and a second downstream primer, or a third upstream primer and a third downstream primer; The nucleotide sequence of the first upstream primer is shown in SEQ ID NO.1; The nucleotide sequence of the first downstream primer is shown in SEQ ID NO.2; The nucleotide sequence of the second upstream primer is shown in SEQ ID NO.3; The nucleotide sequence of the second downstream primer is shown in SEQ ID NO.4; The nucleotide sequence of the third upstream primer is shown in SEQ ID NO.5; The nucleotide sequence of the third downstream primer is shown in SEQ ID NO.6.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This invention uses Brucella BCSP31 and Brucella cspA genes as target genes. By screening for Brucella BCSP31 genes, it can be determined whether an animal is infected with Brucella, and by screening for Brucella cspA genes, it can be determined whether an animal has been vaccinated with the BA0711 vaccine strain.

[0011] More specifically, if the test result of the first kit is positive, it indicates that the animal has been infected with Brucella or has been vaccinated against Brucella. Combining the results of the second kit, if the second kit is negative and the first kit is positive, it indicates that the animal has been vaccinated with the BA0711 vaccine strain and has developed good immune protection; if the second kit is positive and the first kit is positive, it indicates that the animal is infected with Brucella; if both the first and second kits are negative, it indicates that the animal is neither infected with Brucella nor vaccinated against Brucella. Attached Figure Description

[0012] Figure 1 This is the detection result of the first primer set; Figure 2 This is the detection result of the second primer set; Figure 3 This is the detection result of the third primer set; Figure 4 This is the result of testing a flock of sheep using the second primer set. Detailed Implementation

[0013] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0014] Example 1 I. Materials One sample each of sheep spleen tissue, sheep placenta, amniotic fluid, milk, and swab; BA0711 vaccine strain, Brucellamelitensis 16M strain, and Brucella melitensis 2308 strain; Detection reagents: PCR enzyme premix, DL2000 DNA molecular marker, Tiangen DNA extraction kit (DP304). II. Detection Methods 1. PCR detection of Brucella BCSP31 gene (1) Primer source: WS 269—2019 Brucellosis Diagnosis (2) The primer sequences of the first primer set are: Primer B4 5'-TGG CTC GGT TGC CAA TAT CAA-3' (789-809) Primer B5 5'-CGC GCT TGC CTT TCA GGT CTG-3' (1012-992) The amplification product size is 224 bp.

[0015] (3) PCR system: The total volume is 50 µL, including 25 µL of PCR Mix, 1 µL each of upstream and downstream primers, 5 µL of template DNA, and 18 µL of deionized water.

[0016] (4) Reaction conditions: 94 °C pre-denaturation for 4 min, 94 °C denaturation for 1 min, 58 °C annealing for 1 min, 72 °C extension for 1 min, cycled 30 times, and then extended at 72 °C for another 5 min.

[0017] (5) Experimental results reference Figure 1 ; Figure 1 In the diagram, the symbols represent the following: M: DL2000 DNA Ladder; 1: Spleen; 2: Placenta; 3: Milk; 4: Swab; 5: BA0711 vaccine strain; 6: Brucella melitensis 16M; 7: Brucella melitensis 2308; -: Negative control.

[0018] Results: The nucleic acid test results for the Brucella BCSP31 gene in spleen, placenta, milk, swabs, BA0711 vaccine strain, Brucella melitensis 16M, and Brucellamelitensis 2308 samples were all positive.

[0019] 2. Brucella cspA gene PCR detection (1) Primer source: This invention developed two primer sets for detecting the Brucella cspA gene, as follows: ①The sequence of the second primer set is F: 5'-CGGACAGGTCAAATTCTTC-3'; R 5'-GCAGCCTATGTAACAATCC-3', and the amplified fragment size is 307bp.

[0020] ② Sequence of the third primer set: F1: 5'-ATGGCACAGACCGGACAGGTCAAAT-3'; R1 5'-GGATGCGCTCTCATGCAGCCTATG-3', the amplified fragment size is 332bp.

[0021] (3) PCR system: The total volume is 50 µL, including 25 µL of PCR Mix, 1 µL each of upstream and downstream primers, 5 µL of template DNA, and 18 µL of deionized water.

[0022] (4) Reaction conditions: 94 °C pre-denaturation for 4 min, 94 °C denaturation for 1 min, 58 °C annealing for 1 min, 72 °C extension for 1 min, cycled 30 times, and then extended at 72 °C for another 5 min.

[0023] (5) Experimental results reference Figure 2 and Figure 3 ; Figure 2 This is the amplification result of the first primer set; Figure 3 This is the amplification result of the second primer set; Figure 2 and Figure 3 In the diagram, the symbols represent the following: M: DL2000 DNA Ladder; 1: Spleen; 2: Placenta; 3: Milk; 4: Swab; 5: BA0711 vaccine strain; 6: Brucella melitensis 16M; 7: Brucella melitensis 2308; -: Negative control.

[0024] The results showed that the Brucella cspA gene nucleic acid test was negative in spleen, placenta, milk, swabs, and BA0711 vaccine strain, while the Brucella cspA gene nucleic acid test was positive in Brucella melitensis 16M and Brucella melitensis 2308 samples.

[0025] In practical applications, the above primer sets can be flexibly combined for detection; For example, using the first primer set and the second primer set; or, the first primer set and the third primer set; If the first primer set is positive, it indicates that the animal has been infected with Brucella or has been vaccinated against Brucella. Combining the results of the second or third primer set, if the second or third primer set is negative and the first primer set is positive, it indicates that the animal has been vaccinated with the BA0711 vaccine strain and has developed good immune protection. If the second or third primer set is positive and the first primer set is positive, it indicates that the animal is infected with Brucella. If both the first and second or third primer sets are negative, it indicates that the animal is neither infected with Brucella nor vaccinated against Brucella.

[0026] Example 2 The second primer set mentioned above was used to test a flock of sheep at a certain farm; I. Materials Samples submitted for testing: 23 serum samples and 21 secretion samples; Detection reagents: Taq enzyme amplification system mixture, DNA molecular weight marker, Brucella infection antibody detection kit, DNA extraction kit.

[0027] II. Detection Methods 1. PCR: Extract nucleic acid DNA from the sample and amplify the DNA of virulent Brucella bovis and Brucella melitensis in the sample and control using PCR primers GCAGCCTATGTAACAATCC and CGACAGGTCAAATTCTTC. The amplification product of Brucella bovis and Brucella melitensis was 308 BP, while no amplification product was found in strain BA0711. 2. Antibody detection: Perform according to the kit instructions.

[0028] III. Results 1. PCR amplification results: such as Figure 4 As shown, among the 21 secretion samples, except for 67 / 72 / 74, the remaining 18 samples all showed nucleic acid fragments of wild-type strains (not BA0711). Among them, V is the BA0711 control, BA is the bovine strain control, and BM is the sheep strain control.

[0029] 2. Antibody test results: Except for negative results for samples 40 and 96, and suspected results for samples 39 / 45 and 49, all other serum samples showed the presence of antibodies. Please refer to Table 1 for details.

[0030] Table 1 Test Results Positive control 0.175 101.13 Positive control negative control 0.92 7.88 negative control Positive control 0.193 98.87 Positive control negative control 1.046 (7.88) negative control 30 0.494 61.20 Positive infection 38 0.475 63.58 Positive infection 39 0.796 23.40 Suspected 40 1 (2.13) Negative 45 0.826 19.65 Suspected 46 0.709 34.29 Positive infection 48 0.755 28.54 Positive infection 49 0.791 24.03 Suspected 52 0.37 76.72 Positive infection 54 0.731 31.54 Positive infection 59 0.717 33.29 Positive infection 61 0.692 36.42 Positive infection 62 0.778 25.66 Positive infection 67 0.404 72.47 Positive infection 72 0.719 33.04 Positive infection 74 0.676 38.42 Positive infection 75 0.653 41.30 Positive infection 86 0.503 60.08 Positive infection 88 0.729 31.79 Positive infection 89 0.662 40.18 Positive infection 91 0.719 33.04 Positive infection 92 0.746 29.66 Positive infection 96 0.868 14.39 Negative

[0031] Conclusions: 1. The sheep flock has a serious Brucella infection and should be closely monitored; 2. The widespread infection in this flock indicates that the detection accuracy of this invention is high.

[0032] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, nor does it mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A primer set for distinguishing the BA0711 vaccine strain, characterized in that, This includes a first primer pair targeting the Brucella BCSP31 gene and a second primer pair targeting the Brucella cspA gene. The first primer pair includes a first upstream primer and a first downstream primer; The second primer pair includes a second upstream primer and a second downstream primer, or a third upstream primer and a third downstream primer; The nucleotide sequence of the first upstream primer is shown in SEQ ID NO.1; The nucleotide sequence of the first downstream primer is shown in SEQ ID NO.2; The nucleotide sequence of the second upstream primer is shown in SEQ ID NO.3; The nucleotide sequence of the second downstream primer is shown in SEQ ID NO.4; The nucleotide sequence of the third upstream primer is shown in SEQ ID NO.5; The nucleotide sequence of the third downstream primer is shown in SEQ ID NO.

6.

2. The use of the primer set preparation kit as described in claim 1; the kit is used to distinguish between the BA0711 vaccine strain and the Brucella wild-type strain.

3. A reagent kit, characterized in that, Including the first reagent kit and the second reagent kit; The first kit includes a first primer pair; the second kit includes a second primer pair. The first primer pair includes a first upstream primer and a first downstream primer; The second primer pair includes a second upstream primer and a second downstream primer, or a third upstream primer and a third downstream primer; The nucleotide sequence of the first upstream primer is shown in SEQ ID NO.1; The nucleotide sequence of the first downstream primer is shown in SEQ ID NO.2; The nucleotide sequence of the second upstream primer is shown in SEQ ID NO.3; The nucleotide sequence of the second downstream primer is shown in SEQ ID NO.4; The nucleotide sequence of the third upstream primer is shown in SEQ ID NO.5; The nucleotide sequence of the third downstream primer is shown in SEQ ID NO.6.

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