Kit for identifying brucellosis pathogens based on restriction endonuclease coding and multiple rolling circle amplification

Abstract

The invention discloses a kit for identifying brucellosis pathogens based on restriction endonuclease coding and multiple rolling circle amplification, and belongs to the technical field of pathogen detection. The invention designs a target sequence, an RE coding lock-type probe, a rolling circle amplification primer and an asymmetric PCR (Polymerase Chain Reaction) upstream and downstream primer group for actual sample detection, wherein the target sequence, the RE coding lock-type probe and the rolling circle amplification primer are used for detecting bovine breed Brucella, sheep breed Brucella, swine breed Brucella, bovine breed Brucella wild strain 2308 and vaccine strain A19, and then the kit for identifying brucellosis pathogens is obtained. The kit for identifying brucellosis pathogens has the advantages of high sensitivity, strong specificity, no dependence on expensive variable temperature instruments and equipment and capability of completing detection at normal temperature, can accurately realize brucellosis strain type identification and diagnosis, and has important significance on early prevention and control of brucellosis.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogen detection, and in particular relates to a kit for identifying brucellosis pathogens based on restriction endonuclease coding and multiple rolling circle amplification. Background Art

[0002] Brucellosis, also known as Mediterranean fever, Malta fever, or simply brucellosis, is a zoonotic infectious disease caused by bacteria of the genus Brucella. Brucellosis is generally susceptible to livestock, such as cattle, sheep, pigs, and dogs. The clinical manifestations are abortion or infertility in female livestock; male livestock have symptoms such as orchitis. Epidemiological survey results show that the prevalent types of brucellosis in my country are mostly Brucella bovis, Brucella ovis, and Brucella suis, and the proportion of brucellosis caused is as high as 93.21%. It can be seen that the establishment of a rapid and simple detection and identification method for Brucella bovis, Brucella ovis, and Brucella suis is of great significance for the prevention, control, and purification of brucellosis.

[0003] The diagnosis and detection methods of brucellosis have always been an important part of the prevention and control of the disease, mainly including three aspects: etiology, serology and molecular biology detection. Although the isolation and culture of etiology is the "gold standard" for Brucella detection, it is time-consuming and has high operational safety risks. It must be completed by professionals in a BSL-3 laboratory and is not suitable for routine implementation in ordinary institutions. Serological methods are one of the commonly used detection methods, including agglutination tests (for example: tiger red plate agglutination test RBT and test tube agglutination test SAT) and enzyme-linked immunosorbent assay (ELISA). However, it is still difficult to distinguish between species, especially to distinguish between naturally infected and artificially immunized animals. In addition, there are also shortcomings such as many operating steps and low detection efficiency.

[0004] In the field of molecular biology nucleic acid detection technology, sequencing and PCR and its derivative technologies are currently the most commonly used molecular biology methods for pathogen identification, but the above molecular biology methods all rely on expensive variable temperature instruments and equipment and are time-consuming.

[0005] With the demand for more portable and rapid detection equipment and technologies, isothermal amplification technology with greater development space is receiving more and more attention. At present, among the nucleic acid detection technologies for pathogen identification, LAMP (loop-mediated isothermal amplification) technology has been tried, studied and applied a lot. Among the many isothermal amplification technologies with wide selectivity, unique characteristics and complementary advantages, another promising technology is rolling circle amplification (RCA). In 2018, there were reports of using rolling circle amplification technology for DNA barcoding. Rolling circle amplification technology uses circular single-stranded DNA as a template and uses strand displacement DNA polymerase to catalyze the extension of primers to generate a large amount of high molecular weight single-stranded DNA products. Compared with variable temperature amplification technology, the advantages of rolling circle amplification technology are that it does not need to rely on expensive variable temperature equipment, can be carried out at room temperature, is more convenient to operate, has high sensitivity, strong amplification ability, high sequence specificity, and can distinguish different patterns of a single site. RCA has been widely used in cell, DNA, RNA, protein and small molecule detection, but there are no reports on the use of RCA technology for the identification of Brucella species.

[0006] Restriction endonucleases (RE) are a class of endonucleases that can recognize specific nucleic acid sequences and cut DNA at certain sites. In particular, REs of the Type IIP subclass have accurate and stable recognition and cutting characteristics. The rich variety of REs, numerous accurate recognition sequences, diverse cutting sites and large-scale commercial development make this rich molecular recognition information resource usable as a molecular coding information library. Although RCA's padlock probe has its own outstanding advantages in nucleic acid detection, the low-throughput, single-channel mode of one probe detecting one sample still needs to be broken through. Therefore, the multiplex probe encoding strategy has gradually become a solution that is being explored and has great prospects. Summary of the invention

[0007] In view of this, the object of the invention is to provide a kind of test kit based on restriction endonuclease coding and multiple rolling circle amplification to the identification of brucellosis pathogen, the present invention has designed the RCA padlock probe of three restriction endonuclease codings, by optimizing conditions, at normal temperatures, utilize rolling circle amplification technology to detect common Brucella species (cattle species, sheep species and pig species), and wild strain (Brucella.abortus 2308) and vaccine strain (Brucella.abortus A19) are differentiated, realize multiple, high-throughput nucleic acid detection, greatly simplify operating process.There is no report that brucella is detected based on REco-RCA screening system at present.Therefore this technology is applied in the detection of domestic animal brucellosis, is a major breakthrough of this technology in the field of pathogen detection.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The invention provides a kit for identifying the pathogen of brucellosis based on restriction endonuclease coding and multiple rolling circle amplification (REco-RCA). The kit comprises a primer probe group for amplifying a specific target Targ N of Brucella bovis, a primer probe group for amplifying a specific target Targ Y of Brucella melitensis, a primer probe group for amplifying a specific target Targ Z of Brucella suis, a primer probe group for amplifying a specific target Targ 2308 of a wild strain 2308 of Brucella bovis, a primer probe group for amplifying a specific target Targ A19 of a vaccine strain A19 of Brucella bovis, and corresponding Brucella specific targets;

[0010] The primer probe set for amplifying the bovine Brucella specific target Targ N includes an RE-encoded padlock probe Prob N and a rolling circle amplification primer Pri NY, the nucleotide sequence of the padlock probe Prob N is shown in SEQ ID NO.7, and the sequence of the rolling circle amplification primer Pri NY is shown in SEQ ID NO.10;

[0011] The primer probe set for amplifying the Brucella melitensis specific target Targ Y includes an RE-encoded padlock probe Prob Y and a rolling circle amplification primer Pri NY, the nucleotide sequence of the padlock probe Prob Y is shown in SEQ ID NO.8, and the sequence of the rolling circle amplification primer Pri NY is shown in SEQ ID NO.10;

[0012] The primer probe set for amplifying the Brucella suis specific target Targ Z includes an RE-encoded padlock probe Prob Z and a rolling circle amplification primer Pri Z. The nucleotide sequence of the padlock probe Prob Z is shown in SEQ ID NO.9, and the sequence of the rolling circle amplification primer Pri Z is shown in SEQ ID NO.11.

[0013] The primer probe set for amplifying the specific target Targ 2308 of the wild strain 2308 of Brucella bovis includes an RE-encoded padlock probe Prob 2308 and a rolling circle amplification primer Pri NY, the nucleotide sequence of the padlock probe Prob 2308 is shown in SEQ ID NO.32, and the sequence of the rolling circle amplification primer Pri NY is shown in SEQ ID NO.10;

[0014] The primer probe set for amplifying the bovine Brucella vaccine strain A19 specific target Targ A19 includes RE-encoded padlock probe Prob A19 and rolling circle amplification primer Pri NY, the nucleotide sequence of padlock probe Prob A19 is shown in SEQ ID NO.33, and the sequence of rolling circle amplification primer Pri NY is shown in SEQ ID NO.10.

[0015] Based on the above technical solution, further, the 5′ ends of the probes Prob N, Prob Y, Prob Z, Prob 2308, and ProbA19 are all labeled with a phosphate group.

[0016] Based on the above technical scheme, further, the nucleotide sequence of the bovine Brucella specific target Targ N is shown in SEQ ID NO.1, the nucleotide sequence of the melitensis specific target Targ Y is shown in SEQ ID NO.3, the nucleotide sequence of the suis specific target Targ Z is shown in SEQ ID NO.5, the nucleotide sequence of the bovine Brucella wild strain 2308 specific target Targ 2308 is shown in SEQ ID NO.28, and the nucleotide sequence of the bovine Brucella vaccine strain A19 specific target Targ A19 is shown in SEQ ID NO.30.

[0017] Based on the above technical solution, further, the kit includes upstream primers and downstream primers for asymmetric PCR required to obtain specific targets of bovine, ovine and swine Brucella, bovine Brucella vaccine strain A19 and wild strain 2308.

[0018] Based on the above technical scheme, further, the nucleotide sequence of the upstream primer of the asymmetric PCR of Brucella bovis is shown as SEQ ID NO.12, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.13; the nucleotide sequence of the upstream primer of the asymmetric PCR of Brucella melitensis is shown as SEQ ID NO.14, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.15; the nucleotide sequence of the upstream primer of the asymmetric PCR of Brucella suis is shown as SEQ ID NO.16, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.17; the nucleotide sequence of the upstream primer of the asymmetric PCR of Brucella bovis wild strain 2308 and vaccine strain A19 is shown as SEQ ID NO.34, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.35.

[0019] Based on the above technical solution, further, the kit also includes T4 DNA ligase, ExonucleaseI, Exonuclease III, EcoR I, Hind III, Xho I, phi29 DNA polymerase, T4 DNA ligase buffer, phi29 DNA polymerase buffer, dNTPs, sterile water and SYBR Green II.

[0020] The present invention provides a detection method of the above-mentioned kit, comprising the following steps:

[0021] (1) Ligation reaction: 1-2 μL of 10×T4 DNA ligase buffer, 1-2 μL of RE-encoded padlock probe, 1-2 μL of Brucella-specific target, 0.5-1 μL of T4 DNA ligase, add sterile water to 10 μL, react at 36-38°C for 5-15 min, and react at 71-73°C for 5-15 min;

[0022] (2) Exonuclease reaction: Exonuclease I and Exonuclease III are added to the ligation reaction product obtained in step (1) to perform enzymatic digestion on the ligation reaction product to remove excess bases. The system is reacted at 36-38° C. for 20-30 min and at 85-95° C. for 15-25 min.

[0023] (3) RE digestion reaction: add 1-3 μL of restriction endonuclease (EcoR I or Hind III or Xho I) to the exonuclease reaction product obtained in step (2) to digest the “stem” of the RCA template obtained by the exonuclease reaction. The system is reacted at 36-38°C for 10-30 min and at 71-73°C for 5-15 min.

[0024] (4) RCA amplification reaction: RCA reaction system: 4-6 μL of 10× phi 29 DNA polymerase buffer, 2-3 μL of 10 μM dNTPs, 1 μL of 1% SYBR Green II, 1-2 μL of RCA primer, 1-6 μL of RE digestion product obtained in step (3), 1.5-3.5 U of phi29 DNA polymerase, make up to 50 μL with sterile water, react at 35-38° C. for 50-70 min, and detect in real time using a fluorescent quantitative PCR instrument or an enzyme-labeled instrument.

[0025] Based on the above technical solution, further, the concentration of the RE-encoded padlock probe described in step (1) is above 1 μM, the concentration of the Brucella-specific target is 1-10 μM, and the concentration of T4 DNA ligase is 200-500 U / L.

[0026] Based on the above technical solution, further, the amount of Exonuclease I and Exonuclease III added in step (2) is 0.1-1 μL, and the concentration is 5-200 U / μL.

[0027] Based on the above technical solution, further, the amount of restriction endonucleases (EcoR I, HindIII and Xho I) added in step (3) is 0.5-5 μL, and the concentration is 5-200 U / μL.

[0028] Based on the above technical solution, further, the concentration ratio of the rolling circle amplification primer and the RE-encoded padlock probe described in step (4) is ≥1, and the phi29 DNA polymerase is ≥2.5U.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] 1. The present invention uses restriction endonucleases to digest bovine, ovine and swine Brucella templates, and the digested templates are used for RCA amplification. The presence of the amplified product can be determined by the strength or presence of the fluorescent signal, thereby being able to more accurately achieve differential diagnosis of common Brucella species.

[0031] 2. REco-RCA is an isothermal nucleic acid amplification reaction suitable for portable detection equipment, so it is convenient for actual production on-site detection of common Brucella species (bovine, ovine and porcine), as well as wild strain 2308 and vaccine strain A19.

[0032] 3. The kit of the present invention has strong specificity and high detection accuracy. In terms of species-specific detection, it is determined that there is no cross reaction with species of the same family of Brucella (Falsochrobactrum), and it is also determined that there is no cross reaction with bacterial pathogens (Pseudomonas aeruginosa, Escherichia coli), viral pathogens (Hepatitis B virus, Bovine atadenovirus D) and parasitic pathogens (Toxoplasma gondii, Echinococcus granulosus); in addition, in terms of target base mutation specificity, one, two and three base mutation targets are designed for detection, and there is no cross reaction, which is of great significance for the differential diagnosis of livestock diseases.

[0033] 4. The present invention provides a screening method for identifying common Brucella species (bovine, ovine and porcine species), as well as wild strains (Brucella. abortus 2308) and vaccine strains (Brucella. abortus A19) based on restriction endonuclease-encoded multiple rolling circle amplification. Compared with existing methods for differential diagnosis of Brucella, the present invention has the advantages of low detection cost, simple operation, rapid reaction, high sensitivity and specificity, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0035] Figure 1 Schematic diagram of the REco-RCA probe structure of Brucella bovis, Brucella melitensis and Brucella suis in Example 1, wherein (A) is Brucella bovis, (B) is Brucella melitensis, and (C) is Brucella suis.

[0036] Figure 2 This is an optimization diagram of the RCA template concentration in Example 2, wherein the template concentrations are 0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM and a negative control without template.

[0037] Figure 3 This is an optimization diagram of the ratio of RCA template (T) to RCA primer (P) in Example 2, where the ratios of RCA template to RCA primer are 1:0.25, 1:0.5, 1:1 and 1:2, respectively, and a negative control without primers, where the negative control is sterile water.

[0038] Figure 4This is the concentration optimization diagram of phi29 DNA polymerase in Example 2, where the polymerase concentrations are 1.5, 2.0, 2.5, and 3.0 U, a total of 4 concentration gradients of phi29 DNA polymerase and a negative control without phi29 DNA polymerase, and the negative control is sterile water.

[0039] Figure 5 This is the optimization diagram of the RCA template enzyme cleavage time in Example 2, with enzyme cleavage times of 5 min, 10 min, 20 min, and 30 min, respectively, for a total of 4 time periods and a negative control without adding restriction endonuclease, wherein (A) is Brucella bovis, (B) is Brucella melitensis, and (C) is Brucella suis.

[0040] Figure 6 It is the sensitivity amplification graph of REco-RCA detecting Brucella in Example 3, wherein (A) is a fluorescence graph, (B) is a fitting curve graph, and (C) is a linear graph. 4000pM, 2000pM, 1000pM, 400pM, 200pM, 100pM, 40pM, 20pM, 10pM, 4pM, and 10 concentration gradients of targets and sterile water without target were set as a negative control group, with a total of 11 concentrations.

[0041] Figure 7 This is the species-specific amplification diagram of REco-RCA detection of Brucella in Example 4. Seven bacterial species of the same family but different genera, as well as different species and genera as brucella were designed for specific detection.

[0042] Figure 8 The relative fluorescence bar graph of base mutation target specificity of REco-RCA detection of Brucella in Example 4 was designed to perform specific detection on Brucella suis target (Targ Z) with 1 (Targ Z1), 2 (Targ Z2) and 3 base mutation targets (Targ Z3).

[0043] Fig. 9 This is the gel electrophoresis diagram for optimizing the annealing temperature of asymmetric PCR primers in Example 5, wherein: (A) asymmetric PCR primers for Brucella bovis, (B) asymmetric PCR primers for Brucella melitensis, (C) asymmetric PCR primers for Brucella suis, M: DL500 DNA Marker; Lane 1: 40°C; Lane 2: 45°C; Lane 3: 50°C; Lane 4: 55°C; Lane 5: 60°C; Lane 6: 65°C; Lane 7: Blank.

[0044] Fig.10This is a diagram of the optimization of the concentration ratio of upstream and downstream primers in asymmetric PCR in Example 5, wherein (A) is a gel electrophoresis diagram, and (B) is a fluorescence diagram, wherein M: DL500 DNA Marker; Lane 1: 0:1; Lane 2: 1:1; Lane 3: 5:1; Lane 4: 10:1; Lane 5: 15:1; Lane 6: 20:1.

[0045] Fig.11 These are the actual sample DNA detection diagrams of REco-RCA in Example 5, where (A) is the actual sample detection of Brucella bovis, (B) is the actual sample detection of Brucella melitensis, and (C) is the actual sample detection of Brucella suis. The negative control is sterile water.

[0046] Fig.12 This is the plasmid DNA of actual samples of Brucella bovis, Brucella melitensis and Brucella suis detected by qPCR in Example 6.

[0047] Fig.13 Schematic diagram of the REco-RCA probe structure of the wild-type Brucella bovis strain 2308 and the vaccine strain A19 in Example 7, wherein (A) is the wild-type Brucella bovis strain 2308, and (B) is the vaccine strain A19 of Brucella bovis.

[0048] Fig.14 The actual sample detection diagram of REco-RCA in Example 8, wherein (A) is the wild-type Brucella bovis strain 2308, (B) is the Brucella bovis vaccine strain A19, and the negative is the sterile water control.

[0049] Fig.15 This is the actual sample DNA of qPCR detection of bovine Brucella wild strain 2308 and vaccine strain A19 in Example 9. DETAILED DESCRIPTION

[0050] The present invention is described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are all conventional instruments, reagents and materials already available in the prior art, and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. already available in the prior art.

[0051] Example 1 Design of targets, RE-encoded probes, etc. for REco-RCA detection of Brucella bovis, Brucella melitensis and Brucella suis

[0052] 1. Materials: Plasmid DNA of omp2a gene of three species of Brucella bovis, Brucella melitensis and Brucella suis were synthesized by Nanjing GenScript Biotech Co., Ltd. and cloned into vector pUC57.

[0053] 2. Samples and reagents: Oligonucleotides were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.; phi29 DNA polymerase was purchased from Thermofish (USA). SYBR Green II was purchased from Invitrogen (Waltham, MA). T4 DNA ligase, dNTP, 2× Taq Master Mix, EcoR I, Hind III, and Xho I were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). SYBR qPCR Master Mix and PCR product recovery kit were purchased from Novozyme Biotechnology Co., Ltd. (Nanjing, China). DNA Good View TM nucleic acid dye was purchased from Beijing Saibaisheng Gene Technology Co., Ltd. (Beijing, China).

[0054] 3. Target, probe and primer design: The target sequence (SEQ ID NO.1) for detecting Brucella bovis provided by the present invention comes from the omp2a gene of Brucella bovis, and the nucleotide sequence of the omp2a gene is shown in SEQ ID NO.2; the target sequence (SEQ ID NO.3) for detecting Brucella melitensis comes from the omp2a gene of Brucella melitensis, and the nucleotide sequence of the omp2a gene is shown in SEQ ID NO.4; the target sequence (SEQ ID NO.5) for detecting Brucella suis comes from the omp2a gene of Brucella suis, and the nucleotide sequence of the omp2a gene is shown in SEQ ID NO.6.

[0055] Biological sequence analysis software was used to find highly specific sequence segments of the omp2a gene of three species of Brucella bovis, Brucella melitensis and Brucella suis as target nucleic acid sequences for REco-RCA amplification (as shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5). According to the target nucleic acid sequence, RE-encoded probes for RCA amplification were designed, including bovine probe Prob N (sequence as shown in SEQ ID NO.7, with a phosphate group labeled at the 5′ end); ovine probe Prob Y (sequence as shown in SEQ ID NO.8, with a phosphate group labeled at the 5′ end); and porcine probe Prob Z (sequence as shown in SEQ ID NO.9, with a phosphate group labeled at the 5′ end), ensuring that both ends of the probe (5′ end and 3′ end) were complementary to the target sequence bases. At the same time, a nucleotide sequence partially complementary to the RE-encoded padlock probe was designed as a primer for initiating REco-RCA amplification of Brucella bovis, Brucella ovis and Brucella suis, Pri NY as a primer for initiating RCA amplification of Brucella bovis and Brucella ovis (as shown in SEQ ID NO.10); and Pri Z as a primer for initiating RCA amplification of Brucella suis (as shown in SEQ ID NO.11). In the present invention, the coding domains of the RE-coded padlock probes of Brucella bovis, Brucella melitensis and Brucella suis are designed with recognition sequences of restriction endonucleases EcoR I, Hind III and Xho I with specific and efficient cutting, respectively, to encode the probes, and the structures of the RE-coded padlock probes are analyzed and confirmed using nucleic acid structure software (RNA Structure) (see Figure 1 ) to ensure that each probe contains and only contains a unique specific RE recognition sequence.

[0056] The asymmetric PCR primers for the omp2a gene of Brucella bovis were designed using the software PrimerPremier 5.0, which were marked as SEQ ID NO.12 (upstream primer) and SEQ ID NO.13 (downstream primer); the asymmetric PCR primers for the omp2a gene of Brucella melitensis were marked as SEQ ID NO.14 (upstream primer) and SEQ ID NO.15 (downstream primer); the asymmetric PCR primers for the omp2a gene of Brucella suis were marked as SEQ ID NO.16 (upstream primer) and SEQ ID NO.17 (downstream primer).

[0057] Falsochrobactrum (SEQ ID NO.18) belonging to the same family (Brucellaceae) as Brucella and other common pathogenic microorganisms, Pseudomonas aeruginosa (SEQ ID NO.19), Escherichia coli (SEQ ID NO.20), Hepatitis B virus (SEQ ID NO.21), Bovine atadenovirus D (SEQ ID NO.22), Toxoplasma gondii (SEQ ID NO.23), Echinococcus granulosus (SEQ ID NO.24) were found on the NCBI website, and DNA fragments of the above pathogens were synthesized and used as targets for species-specific detection.

[0058] In addition, the present invention takes Brucella suis as an example, and based on its target Targ Z (SEQ ID NO.5), designs a target with 1 base mutation Targ Z1 (SEQ ID NO.25), 2 base mutation targets Targ Z2 (SEQ ID NO.26), and 3 base mutation targets Targ Z3 (SEQ ID NO.27), and the synthetic DNA is used as the target to be tested for base mutation-specific detection.

[0059] Example 2 RCA amplification reaction

[0060] 1. Ligation reaction: 1 μL of 10×T4 DNA ligase buffer, 1 μL of 1 μM RE-encoding probe (Prob N or Prob Y or Prob Z), 1 μL of 1 μM target (Targ N or Targ Y or Targ Z), 1 μL of 350 U / L T4 DNA ligase, and sterile water to 10 μL. The system reacts at 37°C for 10 min and at 72°C for 10 min.

[0061] 2. Exonuclease reaction: Add 5U / μL Exonuclease I and 200U / μL Exonuclease III to the ligation product obtained in step 1 to digest the ligation product and remove the excess bases, i.e., the part that is not formed into a ring during the ligation process between the target and the probe. React at 37°C for 30 minutes and at 90°C for 20 minutes.

[0062] 3. RE digestion reaction: Add 1 μL restriction endonuclease (EcoR I 50U / μL or Hind III 15U / μL or Xho I 10U / μL) to the exonuclease reaction product obtained in step 2 to digest the "stem" of the circular RCA template. After the correct RE digestion, the RCA template will not be able to participate in RCA amplification and no fluorescence value will be generated; the system is reacted at 37°C for 30min and at 72°C for 10min;

[0063] 4. RCA amplification reaction

[0064] RCA reaction system: 10×phi29 DNA polymerase buffer 5μL, 10μmol / L dNTPs 2.5μL, 1% SYBR Green II 1μL, 1μM RCA primer 2μL (Prob N and Prob Y use RCA primer SEQ ID NO.10, Prob Z uses RCA primer SEQ ID NO.11), 4μL of RCA template digested by RE obtained in step 3, 2.5U phi29 DNA polymerase, and sterile water to 50μL. React at 37℃ for 50min, and use Roche LC 480 fluorescence quantitative PCR instrument for real-time detection.

[0065] In order to achieve the best reaction results, the experimental conditions were optimized for RCA template concentration, RCA template to RCA primer ratio, phi29 DNA polymerase concentration and RE enzyme digestion time of RCA template.

[0066] (1) Optimization of RCA template concentration: Taking the Brucella suis RCA template as an example, four RCA template concentrations were set, namely 0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM and a negative control without RCA template (0 μM). The results showed (see Figure 2 ), when the RCA template concentration was 2.0 μM, RCA amplification was significant and the fluorescence signal tended to saturation, and 2.0 μM was selected as the optimal RCA template concentration.

[0067] (2) RCA template and RCA primer ratio optimization: Taking the Brucella suis RCA template as an example, the concentration ratio of the Brucella suis RCA template and the RCA primer Pri Z (SEQ ID NO.11) in the RCA reaction system was adjusted to set the optimized ratios to 1:0.25, 1:0.5, 1:1 and 1:2, respectively, and the negative control was set without RCA primer. The results show (see Figure 3 ), when the ratio of RCA template to RCA primer was 1:1, the fluorescence signal tended to be saturated, and 1:1 was selected as the optimal ratio of RCA template to RCA primer.

[0068] (3) Optimization of phi29 DNA polymerase concentration: phi29 DNA polymerase is an important catalytic enzyme in the RCA reaction system. The concentration of the enzyme has a very important effect on the amplification effect. We designed 1.5U, 2.0U, 2.5U, 3.0U of phi29 DNA polymerase, a total of four concentration gradients, and a negative control without phi29 DNA polymerase (see Figure 4 ), with the increase of phi29 DNA polymerase concentration, the amplification effect became more significant, and the concentration of 2.5U with better amplification effect was selected for subsequent experiments.

[0069] (4) Optimization of the time for RE to cleave the RCA template: After the RCA template is cleaved by the correct RE, rolling circle amplification cannot be performed and no fluorescence value is generated. However, the inability of the incorrect RE to cleave the RCA template does not affect the next step of RCA amplification and fluorescence value is generated. The present invention interprets the detection result by the size of the fluorescence value generated by RCA amplification, so it is particularly important for the present invention that RE completes the cleavage of the RCA template in a short time. The results show (see Figure 5 ), with the increase of enzyme digestion time, the cutting efficiency gradually increased. When the enzyme digestion time was 20 minutes, the cutting rate of RCA template of Brucella bovis, melittis and suis could reach 98.34%, 99.99% and 99.99% respectively. Therefore, 20 minutes was selected as the optimal enzyme digestion time of RCA template of Brucella bovis, melittis and suis, which greatly reduced the detection time.

[0070] Example 3 Detection sensitivity

[0071] Taking Brucella suis as an example, the Brucella suis target Targ Z (SEQ ID NO.5) was diluted step by step to 10 concentration gradients of 4000pM, 2000pM, 1000pM, 400pM, 200pM, 100pM, 40pM, 20pM, 10pM, and 4pM for use, and sterile water without target was used as a negative control group for sensitivity detection.

[0072] (1) Ligation reaction: 1 μL of 10×T4 DNA ligase buffer, 1 μL of 1 μM RE-encoding probe (Prob Z), 1 μL of target (4000 pM or 2000 pM or 1000 pM or 400 pM, 200 pM or 100 pM or 40 pM or 20 pM or 10 pM or 4 pM) (Targ Z), 1 μL of 350 U / L T4 DNA ligase, and sterile water to make up to 10 μL. The system was reacted at 37°C for 10 min and at 72°C for 10 min.

[0073] (2) Exonuclease reaction: Add 5U / μL Exonuclease I and 200U / μL Exonuclease III to the ligation reaction product obtained in step (1) to perform enzyme digestion on the ligation product to remove excess bases, i.e., the uncirculated portion of the target and probe. The reaction was carried out at 37°C for 30 min and at 90°C for 20 min.

[0074] (3) RCA amplification reaction

[0075] RCA reaction system: 5 μL of 10×phi29 DNA polymerase buffer, 2.5 μL of 10 μmol / L dNTPs, 1 μL of 1% SYBR Green II, 2 μL of 1 μM RCA primer (SEQ ID NO.11), 4 μL of Brucella suis RCA template obtained in step (2), 2.5 U of phi29 DNA polymerase, and sterile water to 50 μL. React at 37°C for 50 min, and use Roche LC 480 fluorescence quantitative PCR instrument for real-time detection.

[0076] The experimental results from Figure 6 It can be seen that when the target concentration is 40pM to 2000pM, obvious amplification can be seen. Among them, in the low concentration range (4pM-50pM), the linearity between the detection concentration and the slope is good, and the equation is y=0.006x-0.0021, R 2 =0.9947. The detection limit (LOD) was calculated using the 3δ method and was 2.2811 pM.

[0077] Example 4 Detection specificity

[0078] 1. Species-specific detection. Taking Brucella suis as an example, the sequences of 7 pathogenic microorganisms of the same family but different genera, species and genera as brucella were searched and synthesized: Falsochrobactrum, Pseudomonas aeruginosa, Escherichia coli, Hepatitis B virus, Bovine atadenovirus D, Toxoplasma gondii, Echinococcus granulosus. They were respectively combined with the suis probe Prob Z (SEQ ID NO.9) at a concentration of 100 μM, and the suis target Targ Z (SEQ ID NO.5) was combined with the suis probe Prob Z (SEQ ID NO.9) at a concentration of 1 μM. Sterile water without any target was used as a negative control for the experiment.

[0079] (1) Ligation reaction: 1 μL of 10×T4 DNA ligase buffer, 1 μL of 1 μM RE-encoding probe (Prob Z), 1 μL of 1 μM target (Falsochrobactrum or Pseudomonas aeruginosa or Escherichia coli, Hepatitis B virus or Bovine atadenovirus D or Toxoplasma gondii or Echinococcus granulosus or Targ Z), 1 μL of 350 U / L T4 DNA ligase, and sterile water to make up to 10 μL. The system was reacted at 37°C for 10 min and at 72°C for 10 min.

[0080] (2) Exonuclease reaction: Add 5U / μL Exonuclease I and 200U / μL Exonuclease III to the ligation reaction product obtained in step (1) to perform enzyme digestion on the ligation product to remove excess bases, i.e., the uncirculated portion of the target and probe. The reaction was carried out at 37°C for 30 min and at 90°C for 20 min.

[0081] (3) RCA amplification reaction

[0082] RCA reaction system: 5 μL of 10×phi29 DNA polymerase buffer, 2.5 μL of 10 μmol / L dNTPs, 1 μL of 1% SYBR Green II, 2 μL of 1 μM RCA primer (SEQ ID NO.11), 4 μL of the suis brucella RCA template obtained in step (2), 2.5 U of phi29 DNA polymerase, and sterile water to 50 μL. React at 37°C for 50 min, and use Roche LC 480 fluorescence quantitative PCR instrument for real-time detection.

[0083] The experimental results are as follows Figure 7 As shown, the Brucella suis REco-RCA probe (SEQ ID NO.9) can effectively connect to low concentration (1 μM) of Brucella suis target DNA (SEQ ID NO.5) to form an RCA template, and amplification occurs under the catalysis of phi29 DNA polymerase, while high concentration (100 μM) of the other 7 pathogenic microorganism targets cannot be connected to the Brucella suis REco-RCA probe (SEQ ID NO.9) and cannot be amplified.

[0084] 2. Specific detection of base mutation targets. Taking Brucella suis as an example, one base mutation target (SEQ ID NO.25), two base mutation targets (SEQ ID NO.26), and three base mutation targets (SEQ ID NO.27) were designed based on the target DNA of Brucella suis (SEQ ID NO.5), and they were respectively combined with the porcine probe Prob Z (SEQ ID NO.9) at a concentration of 100 μM. The porcine target Targ Z (SEQ ID NO.5) was combined with the porcine probe Prob Z (SEQ ID NO.9) at a concentration of 1 μM, and sterile water without any target was used as a negative control for the experiment.

[0085] (1) Ligation reaction: 1 μL of 10×T4 DNA ligase buffer, 1 μL of 1 μM RE-encoding probe (Prob Z), 1 μL of 1 μM target (Targ Z1 or Targ Z2 or Targ Z3 or Targ Z), 1 μL of 350 U / L T4 DNA ligase, and sterile water to 10 μL. The system was reacted at 37°C for 10 min and at 72°C for 10 min.

[0086] (2) Exonuclease reaction: Add 5U / μL Exonuclease I and 200U / μL Exonuclease III to the product obtained from the ligation reaction in step (1) to perform enzyme digestion on the ligation product to remove excess bases, i.e., the uncirculated portion of the target and probe. The reaction was carried out at 37°C for 30 min and at 90°C for 20 min.

[0087] (3) RCA amplification reaction

[0088] RCA reaction system: 10×phi29 DNA polymerase buffer 5μL, 10μmol / L dNTPs 2.5μL, 1% SYBR Green II 1μL, 1μM primer (SEQ ID NO.11) 2μL, RCA template 4μL obtained in step (2), 2.5Uphi29 DNA polymerase, sterile water to 50μL. React at 37℃ for 50min, and use Roche LC 480 fluorescence quantitative PCR instrument for real-time detection.

[0089] The results are as follows Figure 8 As shown, the Brucella suis REco-RCA probe (SEQ ID NO.9) can effectively connect and amplify low concentration (1 μM) of Brucella suis target DNA (SEQ ID NO.5) to produce fluorescence values, while high concentration (100 μM) of three base mutation targets cannot be connected to it and no fluorescence values ​​are produced.

[0090] Example 5 Detection of actual samples of Brucella bovis, melittis and swine

[0091] (1) Using the asymmetric PCR method, the annealing temperature and the ratio of upstream and downstream primers were optimized. The asymmetric PCR reaction system was: 12.5 μL 2×Taq Master Mix; 0.5 μL of upstream and downstream primers; 2 μL DNA template, supplemented with sterile water to 25 μL. The asymmetric PCR reaction program was: 94°C for 10 min; 94°C for 30 s, 50°C for 30 s, 72°C for 30 s, 30 cycles; and finally 72°C for 10 min extension.

[0092] In order to ensure that the primers anneal effectively after the asymmetric PCR template is denatured and specifically bind to the template, it is critical to screen the appropriate annealing temperature. The experiment set up 6 annealing temperature gradients of 40℃, 45℃, 50℃, 55℃, 60℃, and 65℃. The results are shown in Fig. 9 , the five annealing temperatures for the Brucella bovis, Brucella melitensis and Brucella suis primers all yielded single bright bands, and the positions were consistent with the expected sizes. The Brucella bovis primers (SEQ ID NO.12 and SEQ ID NO.13) were brighter at an annealing temperature of 50°C; the Brucella melitensis primers (SEQ ID NO.14 and SEQ ID NO.15) were brighter at 40°C, 45°C, 50°C, 55°C, 60°C and 65°C; the Brucella suis primers (SEQ ID NO.16 and SEQ ID NO.17) were brighter at annealing temperatures of 40°C, 45°C, 50°C, 55°C and 60°C. Therefore, the present invention selects 50°C as the annealing temperature for asymmetric PCR primers for Brucella bovis, Brucella melitensis and Brucella suis.

[0093] Taking the Brucella suis primers (SEQ ID NO.16 and SEQ ID NO.17) as an example, different concentrations of upstream and downstream primer ratios (1:1, 5:1, 10:1, 15:1, 20:1) were set. The results are as follows: Fig.10 -A shows that with the increase of upstream primer concentration, the single-stranded DNA product of Brucella suis gradually increased. When the ratio was 20:1, the single-stranded product increased significantly. Fig.10 -B fluorescence image was further confirmed.

[0094] (2) Perform REco-RCA actual sample detection based on the above asymmetric PCR optimization results. First, perform asymmetric PCR amplification on the Brucella bovis, ovine and swine DNA, and use the obtained amplification products as their respective target DNAs to participate in the ligation reaction and exonuclease reaction in Example 2. After the reaction, use different REs (EcoR I, Hind III, Xho I) to perform RE enzyme digestion on the obtained RCA templates of Brucella bovis, ovine and swine. The products obtained after RE enzyme digestion are used as RCA templates for REco-RCA amplification, thereby realizing actual sample detection of Brucella bovis, ovine and swine. The results are shown in the figure. Fig.11 As shown, the RCA template of Brucella bovis cannot participate in rolling circle amplification after being digested by EcoR I; the RCA template of Brucella melitensis cannot participate in rolling circle amplification after being digested by Hind III; the RCA template of Brucella suis cannot participate in rolling circle amplification after being digested by Xho I, and no fluorescence value is generated. Similarly, the inability of incorrect RE to digest the RCA template does not affect its participation in rolling circle amplification, and the fluorescence value is not significantly different from the control group without restriction endonuclease. Therefore, the actual samples of Brucella bovis, Brucella melitensis and Brucella suis can be screened by restriction endonucleases.

[0095] Example 6 qPCR method for detection of Brucella bovis, Brucella melitensis and Brucella suis

[0096] Using bovine, ovine and swine Brucella plasmid DNA as templates, the fluorescent quantitative PCR method was used for detection. The reaction system is: 10μL 2×ChamQ Universal SYBR qPCR Master Mix, 2μL DNA template (bovine or ovine or swine Brucella plasmid DNA), 0.4μL upstream primer (bovine: SEQ ID NO.12; ovine: SEQ ID NO.14; swine: SEQ ID NO.16), 0.4μL downstream primer (bovine: SEQ ID NO.13; ovine: SEQ ID NO.15; swine: SEQID NO.17), and sterile water is added to 20μL. Reaction conditions: 94℃5min; 94℃10s, 55℃30s, 72℃30s; 95℃15s, 55℃1min, 95℃15s, a total of 40 cycles. The results are as follows Fig.12 As shown in the figure, the real-time fluorescence quantitative PCR amplification curve reflects the exponential phase and plateau phase of plasmid DNA amplification of actual samples of Brucella bovis, ovine and swine. The test results verify that the REco-RCA detection method is authentic and reliable for the detection of Brucella bovis, ovine and swine.

[0097] Example 7 Design of targets, RE-encoded probes, etc. for detecting wild-type Brucella bovis strain 2308 and vaccine strain A19 by REco-RCA

[0098] 1. Materials: The inactivated vaccine of B. abortus A19 (abbreviated as A19) was purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The DNA of Brucella vaccine strain A19 was extracted using a viral genomic RNA / DNA extraction kit. The DNA sample of wild strain B. abortus 2308 was preserved by the Key Laboratory of Zoonoses, College of Animal Science and Medicine, Shenyang Agricultural University.

[0099] 2. Samples and reagents: Oligonucleotides were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.; phi29 DNA polymerase was purchased from Thermofish (USA). SYBR Green II was purchased from Invitrogen (Waltham, MA). T4 DNA ligase, dNTP, 2× Taq Master Mix, Hind III, Xho I, and viral genomic RNA / DNA extraction kit were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). SYBR qPCR Master Mix and PCR product recovery kit were purchased from Novozyme Biotechnology Co., Ltd. (Nanjing, China). DNA Good View TM nucleic acid dye was purchased from Beijing Saibaisheng Gene Technology Co., Ltd. (Beijing, China).

[0100] 1. Target, probe and primer design

[0101] The target sequence (SEQ ID NO.28) for detecting bovine Brucella wild strain 2308 provided by the present invention comes from the Ery gene of bovine Brucella wild strain 2308, and the nucleotide sequence of the Ery gene of bovine Brucella wild strain 2308 is shown in SEQ ID NO.29; the target sequence (SEQ ID NO.30) for detecting bovine Brucella vaccine strain A19 comes from the Ery gene of bovine Brucella vaccine strain A19, and the nucleotide sequence of the Ery gene of Brucella vaccine strain A19 is shown in SEQ ID NO.31.

[0102] Biological sequence analysis software was used to compare and search for sequence segments with strong specificity of the Ery gene of wild strain 2308 of Brucella bovis and vaccine strain A19, which were used for the target DNA sequences of REco-RCA (as shown in SEQ ID NO.28 and SEQ ID NO.30). According to the target DNA sequence, RE-coded padlock probes for rolling circle amplification were designed, including wild strain 2308 probe Prob 2308 (as shown in SEQ ID NO.32, with a phosphate group labeled at the 5′ end); vaccine strain A19 probe Prob A19 (as shown in SEQ ID NO.33, with a phosphate group labeled at the 5′ end), and both ends of the probe (5′ end and 3′ end) were ensured to be complementary to the bases of the respective target sequences. At the same time, a nucleotide sequence partially complementary to the RE-coded padlock probe was designed as an RCA primer for initiating the amplification of wild strain 2308 of Brucella bovis and vaccine strain A19, and the primer was named Pri NY (as shown in SEQ ID NO.10). In the present invention, the coding domains of the RE-encoded padlock probes of the wild strain 2308 of Brucella bovis and the vaccine strain A19 are respectively designed with recognition sequences of the restriction endonucleases Xho I and Hind III with specific and efficient cutting, and the probes are encoded. The structures of the RE-encoded padlock probes are analyzed and confirmed using the nucleic acid structure software (RNA Structure) (see Fig.13 ) to ensure that each probe contains and only contains a unique specific RE recognition sequence.

[0103] Asymmetric PCR primers for the Ery gene of Brucella wild strain 2308 and vaccine strain A19 were designed using the software PrimerPremier 5.0, marked as SEQ ID NO.34 (upstream primer) and SEQ ID NO.35 (downstream primer).

[0104] Example 8 Actual sample detection of wild strain 2308 and vaccine strain A19 of Brucella bovis

[0105] Extract the bovine Brucella vaccine strain A19 DNA according to the instructions of the viral genome RNA / DNA extraction kit. After testing, the OD value of the extracted bovine Brucella vaccine strain A19 DNA was 260 / OD 280 The ratios are 1.92 and 1.93 respectively, and the ratio is between 1.8 and 2.0, indicating high purity and can be used for subsequent experimental operations.

[0106] The DNA of wild-type Brucella bovis strain 2308 and vaccine strain A19 was obtained by asymmetric PCR. The reaction system and reaction procedure of asymmetric PCR were the same as those in Example 5. The upstream and downstream primers used in asymmetric PCR of wild-type Brucella bovis strain 2308 and vaccine strain A19 were the same pair of primers (SEQ ID NO.34 and SEQ ID NO.35).

[0107] The bovine wild strain 2308 and vaccine strain A19 products obtained after the above asymmetric PCR amplification were used as targets and combined with the corresponding bovine wild strain 2308 and vaccine strain A19 REco-RCA probes, and ligation reaction, exonuclease reaction, RE enzyme cleavage reaction (Xho I or Hind III) and REco-RCA amplification reaction were performed in sequence. The reaction system, reaction time and reaction temperature of the above four reactions were the same as those in Example 5. The REco-RCA screening technology was used to detect the actual samples of bovine Brucella wild strain 2308 and vaccine strain A19 as shown in Figure 5. Fig.14 As shown, the RCA template of the wild strain 2308 of Brucella bovis cannot participate in RCA amplification after being digested by Xho I, and no fluorescence value is generated; the RCA template of the vaccine strain A19D of Brucella bovis cannot participate in RCA amplification after being digested by Hind III, and no fluorescence value is generated. Similarly, the incorrect restriction endonuclease cannot digest the RCA template, does not affect its RCA amplification, and the fluorescence value is not significantly different from the control group without restriction endonuclease. Therefore, the actual samples of Brucella wild strain 2308 and vaccine strain A19 can be screened by restriction endonuclease.

[0108] Example 9 qPCR method for detection of actual samples of wild strains of Brucella bovis 2308 and A19

[0109] Actual sample DNA of wild strain 2308 of Brucella bovis and vaccine strain A19 were used as templates and detected by fluorescence quantitative PCR. The reaction system was: 10 μL 2×ChamQ Universal SYBR qPCR Master Mix, 2 μL DNA template, 0.4 μL upstream primer (SEQ ID NO.32), 0.4 μL downstream primer (SEQ ID NO.33), and sterile water was added to 20 μL. Reaction conditions: 94℃5min; 94℃10s, 55℃30s, 72℃30s; 95℃15s, 55℃1min, 95℃15s, a total of 40 cycles. The results are as follows Fig.15As shown, the qPCR amplification curve reflects the exponential phase and plateau phase of DNA amplification of actual samples of Brucella wild strain 2308 and vaccine strain A19. The real-time fluorescence quantitative PCR detection results indirectly verify that the detection results of REco-RCA screening technology for bovine Brucella wild strain 2308 and vaccine strain A19 are true and reliable.

[0110] In summary, the present invention establishes REco-RCA screening technology for the first time on the basis of rolling circle amplification technology (one of the nucleic acid constant temperature amplification detection methods), and uses this technology to realize the screening of common types of Brucella. This technology has the advantages of not relying on expensive variable temperature nucleic acid amplification instruments such as RCA, realizing isothermal (37°C) detection, and is particularly suitable for on-site detection in large-scale livestock farms. Sensitivity experiments show that the detection limit of this technology for Brucella is 2.2811pM, which can achieve sensitive detection effects. Specificity experiments show that the target and REco-RCA probe designed by the present invention have good specificity in the differential diagnosis of Brucella species, and have no cross-reaction to other common bacteria, viruses and parasites. In addition, this detection technology shows excellent detection ability. Moreover, the screening technology of the present invention can be completed within 50 minutes, realizing the need for rapid detection and greatly improving the detection efficiency. This provides a more convenient technical means for the detection of bacterial pathogens, and also provides a new technical title for the early pathogen differential diagnosis of brucellosis, and has great application potential in the field of livestock pathogen detection and pathogen diagnosis.

Claims

1. A kit for identifying the pathogen of brucellosis based on restriction endonuclease coding and multiple rolling circle amplification, characterized in that: The kit comprises a primer probe set for amplifying a specific target Targ N of Brucella bovis, a primer probe set for amplifying a specific target Targ Y of Brucella melitensis, a primer probe set for amplifying a specific target Targ Z of Brucella suis, a primer probe set for amplifying a specific target Targ 2308 of wild strain 2308 of Brucella bovis, and a primer probe set for amplifying a specific target TargA19 of vaccine strain A19 of Brucella bovis, as well as corresponding Brucella specific targets; The primer probe set for amplifying the bovine Brucella specific target Targ N includes an RE-encoded padlock probe Prob N and a rolling circle amplification primer PriNY, the nucleotide sequence of the padlock probe Prob N is shown in SEQ ID NO.7, and the sequence of the rolling circle amplification primer PriNY is shown in SEQ ID NO.10; The primer probe set for amplifying the Brucella melitensis specific target Targ Y includes an RE-encoded padlock probe Prob Y and a rolling circle amplification primer PriNY, the nucleotide sequence of the padlock probe Prob Y is shown in SEQ ID NO.8, and the sequence of the rolling circle amplification primer PriNY is shown in SEQ ID NO.10; The primer probe set for amplifying the Brucella suis specific target Targ Z includes an RE-encoded padlock probe Prob Z and a rolling circle amplification primer PriZ. The nucleotide sequence of the padlock probe Prob Z is shown in SEQ ID NO.9, and the sequence of the rolling circle amplification primer PriZ is shown in SEQ ID NO.

11. The primer probe set for amplifying the specific target Targ 2308 of the wild strain 2308 of Brucella bovis includes an RE-encoded padlock probe Prob 2308 and a rolling circle amplification primer PriNY, the nucleotide sequence of the padlock probe Prob 2308 is shown in SEQ ID NO.32, and the sequence of the rolling circle amplification primer PriNY is shown in SEQ ID NO.10; The primer probe set for amplifying the bovine Brucella vaccine strain A19 specific target Targ A19 includes an RE-encoded padlock probe Prob A19 and a rolling circle amplification primer PriNY. The nucleotide sequence of the padlock probe Prob A19 is shown in SEQ ID NO.33, and the sequence of the rolling circle amplification primer PriNY is shown in SEQ ID NO.

10.

2. The kit according to claim 1, characterized in that The 5′ ends of the probes Prob N, Prob Y, Prob Z, Prob2308, and Prob A19 are all labeled with a phosphate group.

3. The kit according to claim 1, characterized in that The nucleotide sequence of the bovine Brucella specific target Targ N is shown in SEQ ID NO.1, the nucleotide sequence of the melitensis specific target Targ Y is shown in SEQ ID NO.3, the nucleotide sequence of the suis specific target Targ Z is shown in SEQ ID NO.5, the nucleotide sequence of the bovine Brucella wild strain 2308 specific target Targ 2308 is shown in SEQ ID NO.28, and the nucleotide sequence of the bovine Brucella vaccine strain A19 specific target Targ A19 is shown in SEQ ID NO.

30.

4. The kit according to claim 1, characterized in that The kit comprises upstream primers and downstream primers for asymmetric PCR required for obtaining specific targets of bovine, ovine and swine Brucella, bovine Brucella vaccine strain A19 and wild strain 2308.

5. The kit according to claim 4, characterized in that The nucleotide sequence of the upstream primer of asymmetric PCR of Brucella bovis is shown as SEQ ID NO.12, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.13; the nucleotide sequence of the upstream primer of asymmetric PCR of Brucella melitensis is shown as SEQ ID NO.14, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.15; the nucleotide sequence of the upstream primer of asymmetric PCR of Brucella suis is shown as SEQ ID NO.16, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.17; the nucleotide sequence of the upstream primer of asymmetric PCR of Brucella bovis wild strain 2308 and vaccine strain A19 is shown as SEQ ID NO.34, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.

35.

6. The kit according to claim 1, characterized in that The kit also includes T4 DNA ligase, Exonuclease I, Exonuclease III, EcoR I, Hind III, Xho I, phi29 DNA polymerase, T4 DNA ligase buffer, phi29 DNA polymerase buffer, dNTPs, sterile water and SYBR Green II.

7. The detection method of the kit according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Ligation reaction: 1-2 μL of 10×T4 DNA ligase buffer, 1-2 μL of RE-encoded padlock probe, 1-2 μL of Brucella-specific target, 0.5-1 μL of T4 DNA ligase, and add sterile water to 10 μL. The system is reacted at 36-38°C for 5-15 min and at 71-73°C for 5-15 min. (2) Exonuclease reaction: Exonuclease I and Exonuclease III are added to the ligation reaction product obtained in step (1) to perform enzymatic digestion on the ligation reaction product to remove excess bases. The system is reacted at 36-38° C. for 20-30 min and at 85-95° C. for 15-25 min. (3) RE digestion reaction: 1-3 μL of restriction endonuclease (EcoR I 50 U / μL, Hind III 15 U / μL or Xho I 10 U / μL) was added to the exonuclease reaction product obtained in step (2) to digest the "stem" of the RCA template obtained by exonuclease reaction. The system was reacted at 36-38°C for 10-30 min and at 71-73°C for 5-15 min. (4) RCA amplification reaction: RCA reaction system: 4-6 μL of 10× phi 29 DNA polymerase buffer, 2-3 μL of 10 μM dNTPs, 1 μL of 1% SYBR Green II, 1-2 μL of RCA primer, 1-6 μL of RE digestion product obtained in step (3), phi29 DNA polymerase, and sterile water to 50 μL. React at 35-38°C for 50-70 min, and use a fluorescent quantitative PCR instrument or an enzyme-labeled instrument for real-time detection.

8. The detection method according to claim 7, characterized in that: The concentration of the RE-encoded padlock probe described in step (1) is above 1 μM, the concentration of the Brucella-specific target is 1-10 μM, and the concentration of T4 DNA ligase is 200-500 U / L.

9. The detection method according to claim 7, characterized in that: The amount of Exonuclease I and Exonuclease III added in step (2) is 0.1-1 μL, and the concentration is 5-200 U / μL; the amount of restriction endonucleases (EcoR I, Hind III and Xho I) added in step (3) is 0.5-5 μL, and the concentration is 5-200 U / μL.

10. The detection method according to claim 7, characterized in that: The concentration ratio of the rolling circle amplification primer to the RE-encoded padlock probe in step (4) is ≥1, and the phi29 DNA polymerase is ≥2.5U.

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