阅读说明：本技术 一种用于检测布鲁氏菌抗原的免疫磁珠试剂盒 (Immunomagnetic bead kit for detecting brucella antigen ) 是由 尚佑军 孙晶晶 曹小安 吴锦艳 尹双辉 周建华 杨顺利 兰喜 李学瑞 刘永生 于 2019-11-11 设计创作，主要内容包括：本发明涉及一种用于检测布鲁氏菌抗原的免疫磁珠试剂盒,属于生物技术领域。所述试剂盒包括：免疫磁珠、qPCR反应混合液、检测布鲁氏菌的上游引物、下游引物和探针；所述免疫磁珠为鼠抗布鲁氏菌M5抗原单克隆IgG抗体与磁珠的偶联物。本发明所述试剂盒灵敏度高、稳定性好,快速有效,与传统分离方法相比,克服了布鲁氏菌病料检出率低以及直扩抗原检测过程中布鲁氏菌病原易降解,且病原DNA提取过程中使用的待检液体体积有限导致所提取的DNA无法达到qPCR检测的最低要求。(The invention relates to an immunomagnetic bead kit for detecting brucella antigen, belonging to the technical field of biology. The kit comprises: immunomagnetic beads, qPCR reaction mixed liquor, upstream primers, downstream primers and probes for detecting Brucella; the immunomagnetic beads are conjugates of mouse brucella resistance M5 antigen monoclonal IgG antibodies and magnetic beads. The kit disclosed by the invention is high in sensitivity, good in stability, rapid and effective, and compared with the traditional separation method, the kit overcomes the defects that the detection rate of brucella pathogens is low, brucella pathogens are easy to degrade in the direct-amplification antigen detection process, and the extracted DNA cannot meet the minimum requirement of qPCR detection due to the limited volume of liquid to be detected used in the pathogen DNA extraction process.)

1. An immunomagnetic bead kit for detecting brucella, the kit comprising: immunomagnetic beads, qPCR reaction mixed liquor, upstream primers, downstream primers and probes for detecting Brucella; the immunomagnetic beads are conjugates of mouse brucella resistance M5 antigen monoclonal IgG antibodies and the magnetic beads; the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, and the probe is a substance of which the 5 'end of the nucleotide sequence is marked with FAM and the 3' end is marked with BHQ1, which are shown as SEQ ID NO. 3.

2. The immunomagnetic bead kit of claim 1, wherein the kit further comprises a PBS buffer.

3. The immunomagnetic bead kit of claim 1, wherein the immunomagnetic beads are stored in the form of an immunomagnetic bead suspension, and the concentration of the immunomagnetic beads in the immunomagnetic bead suspension is 1 mg/mL; the solvent of the immunomagnetic bead suspension comprises PBS buffer.

4. The immunomagnetic bead kit of claim 1, wherein the immunomagnetic bead is prepared by the following steps:

1) washing and resuspending the magnetic beads to obtain washed and resuspended magnetic beads;

2) mixing excessive mouse anti-brucella M5 antigen monoclonal IgG antibody with the washed and resuspended magnetic beads obtained in the step 1), and shaking for 15-60 min at 37 ℃ to obtain an immunomagnetic bead suspension;

3) and 2) carrying out magnetic adsorption on the immunomagnetic bead suspension obtained in the step 2), removing the supernatant, washing and resuspending to obtain the immunomagnetic beads.

5. The immunomagnetic bead kit of claim 4, wherein the mass ratio of the monoclonal IgG antibody against Brucella M5 antigen in step 2) to the magnetic bead in step 1) is 100 μ g/6mg or more.

6. The immunomagnetic bead kit of claim 4, wherein the washing and resuspending of step 1) and step 3) are independently performed using PBS buffer; the PBS buffer was 10mM PBS buffer at pH 7.4.

7. The immunomagnetic bead kit according to any one of claims 1 to 6, wherein the use method of the immunomagnetic bead kit comprises the following steps:

(1) mixing immunomagnetic beads and a sample to be detected in a centrifuge tube, and carrying out oscillation reaction at 37 ℃ for 20-60 min;

(2) performing magnetic adsorption, removing the supernatant, washing and resuspending to obtain brucella-enriched immunomagnetic bead resuspension;

(3) processing the immune magnetic bead resuspension enriched with the Brucella in the step (2) by a thermal cracking method to obtain released Brucella DNA;

(4) mixing the Brucella DNA released in the step (3) with qPCR reaction mixed liquor, upstream primers, downstream primers and probes for detecting Brucella, and performing qPCR amplification;

(5) when the CT value is less than 28, the brucella is judged to be positive, and when the CT value is more than or equal to 28, the brucella is judged to be negative.

8. The immunomagnetic bead kit according to claim 7, wherein the mixing volume ratio of the sample to be tested and the immunomagnetic beads in step (1) is 4-6.

9. The immunomagnetic bead kit of claim 7, wherein the reaction procedure of the thermal cracking process of step (3) is as follows: cracking at 95 deg.C for 10min, and ice-cooling for 3 min.

10. The immunomagnetic bead kit of claim 7, wherein the reaction system of qPCR amplification in step (4) comprises, per 20 μ L:probe qPCR Supermix 10. mu.L; 5 mu L of DNA template; RNase freedH₂O2.6 mu L; upstream primer 0.8 μ L; 0.8 mu L of downstream primer and 0.8 mu L of probe primer;

the reaction procedure for the qPCR amplification was: 5min at 95 ℃; denaturation at 95 ℃ for 10s and renaturation at 60 ℃ for 30s for 40 cycles; storing at 4 ℃.

Technical Field

The invention relates to the technical field of biology, in particular to an immunomagnetic bead kit for detecting brucella antigens.

Background

Brucella is a facultative intracellular parasitic bacterium of gram-negative bacteria, has unique intracellular survival and immune mechanisms, and is a natural epidemic disease. The brucella is not only widely distributed, but also can be parasitized among a plurality of animal hosts, and can be infected to human hosts through animals, so that the human is infected with brucellosis, and the brucella is an allergic zoonosis which seriously harms human and animal health. Brucellosis can cause animal infection through various ways such as skin mucosa, digestive tract and vertical transmission, wherein the most dangerous infection source is pregnant animals suffering from diseases. Brucella can be associated with the expulsion of foetus, amniotic fluid and placenta during delivery or abortion, and milk is also one of the important sources of infection. The brucella mainly infects animals such as cattle, sheep, pigs, dogs and the like. Currently, the genus brucella is classified into 6 types of 19 biotypes according to differences in infected animals and differences in antigenicity, i.e., brucella melitensis (b.melitensis, 3 biotypes), brucella bovis (b.abortus, 8 biotypes), brucella suis (b.suis, 5 biotypes), brucella epididymides (b.ovis, 1 biotype), brucella suis sarrini (b.neotomae, 1 biotype), and brucella canis (b.caris, 1 biotype), by a typing method disclosed by WHO in 1985. Bortg isolated from marine mammals such as whales, dolphins, seals, etc. in 2007, brucella cetacea (b.cetii) and brucella finnii (b.pinipendialis) were isolated. Brucella species (b.microti) were found again in 2008. At present, Brucella abortus, Brucella melitensis and canis are pathogenic to human beings, and the pathogenicity of the Brucella abortus, melitensis and canis are different, so that the clinical significance of three infections of sheep, cattle and pig is the greatest, and the pathogenicity of the sheep is the strongest. In addition, the brucella can generate interference phenomenon in the process of infecting animals and human beings, and because the brucella has a plurality of types and different virulence, the interference phenomenon can occur in the parasitism process, thereby bringing great challenge to the prevention and control of brucellosis.

At present, the conventional detection and diagnosis methods aiming at the brucella infection are many, and the pathogenic diagnosis detection methods comprise: sample detection, enrichment culture, separation culture, animal test, staining microscopy, agglutination reaction, biochemical test, staining test, PCR test and the like. The brucella is slow in growth, high in nutrition requirement, easy to pollute sundry bacteria in the culture process, and greatly influenced by factors such as culture environment, operator technique and the like, so that the separation rate is low, the separation difficulty is high, the operation is complicated, the time consumption is long, certain biological safety problems exist, the brucella is not suitable for rapid diagnosis and treatment of a large number of samples, most tissue samples are required to be in a suspension shape through grinding, the bacterial concentration in the samples is diluted to a certain degree, and other components in the tissues can interfere with the experimental result, so that the false negative detection result is caused. Therefore, it is necessary to research and develop a highly sensitive brucella detection technique.

Disclosure of Invention

The invention aims to provide an immunomagnetic bead kit for detecting brucella antigens. The kit realizes that the lowest limit of the detection of the brucella is 10⁵CFU/mL, negative to other bacteria detection, no cross reaction, and repeat coefficient of variation within the group of 0.97% -1.23%, repeat coefficient of variation between groups of 0.24% -1.23%, have better repeatability.

The invention provides an immunomagnetic bead kit for detecting Brucella, which comprises: immunomagnetic beads, qPCR reaction mixed liquor, upstream primers, downstream primers and probes for detecting Brucella; the immunomagnetic beads are conjugates of mouse brucella resistance M5 antigen monoclonal IgG antibodies and the magnetic beads; the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, and the probe is a substance of which the 5 'end of the nucleotide sequence is marked with FAM and the 3' end is marked with BHQ1, and is shown as SEQ ID NO. 3.

Preferably, the kit further comprises a PBS buffer.

Preferably, the immunomagnetic beads are stored in the form of immunomagnetic bead suspension, and the concentration of the immunomagnetic beads in the immunomagnetic bead suspension is 1 mg/mL; the solvent of the immunomagnetic bead suspension comprises PBS buffer.

Preferably, the preparation method of the immunomagnetic beads comprises the following steps:

1) washing and resuspending the magnetic beads to obtain washed and resuspended magnetic beads;

2) mixing excessive mouse anti-brucella M5 antigen monoclonal IgG antibody with the washed and resuspended magnetic beads obtained in the step 1), and shaking for 15-60 min at 37 ℃ to obtain an immunomagnetic bead suspension;

3) and 2) carrying out magnetic adsorption on the immunomagnetic bead suspension obtained in the step 2), removing the supernatant, washing and resuspending to obtain the immunomagnetic beads.

Preferably, the mass ratio of the monoclonal IgG antibody against Brucella M5 antigen in step 2) to the magnetic beads in step 1) is 100. mu.g/6 mg or more.

Preferably, the washing and resuspending of steps 1) and 3) uses PBS buffer; the PBS buffer was 10mM PBS buffer at pH 7.4.

Preferably, the method for using the immunomagnetic bead kit comprises the following steps:

(1) mixing immunomagnetic beads and a sample to be detected in a centrifuge tube, and carrying out oscillation reaction at 37 ℃ for 20-60 min;

(2) performing magnetic adsorption, removing the supernatant, washing and resuspending to obtain brucella-enriched immunomagnetic bead resuspension;

(3) processing the immune magnetic bead resuspension enriched with the Brucella in the step (2) by a thermal cracking method to obtain released Brucella DNA;

(4) mixing the Brucella DNA released in the step (3) with qPCR reaction mixed liquor, upstream primers, downstream primers and probes for detecting Brucella, and performing qPCR amplification;

(5) when the CT value is less than 28, the brucella is judged to be positive, and when the CT value is more than or equal to 28, the brucella is judged to be negative.

Preferably, the mixing volume ratio of the sample to be tested and the immunomagnetic beads in the step (1) is 4-6.

Preferably, the reaction procedure of the thermal cracking method in the step (3) is as follows: cracking at 95 deg.C for 10min, and ice-cooling for 3 min.

Preferably, the reaction system for qPCR amplification in step (4) comprises, per 20 μ L:

ProbeqPCR Supermix 10. mu.L; 5 mu L of DNA template; RNase Free dH₂O2.6 mu L; upstream primer 0.8 μ L; 0.8 mu L of downstream primer and 0.8 mu L of probe primer;

the reaction procedure for the qPCR amplification was: 5min at 95 ℃; denaturation at 95 ℃ for 10s and renaturation at 60 ℃ for 30s for 40 cycles; storing at 4 ℃.

The invention provides an immunomagnetic bead kit for detecting brucella antigen. The kit can solve the problem that in the prior art, the brucella antigen is easy to degrade in the brucella antigen detection process, and the extracted DNA cannot meet the minimum requirement of qPCR detection due to the limited volume of the venom to be detected in the pathogen DNA extraction process, so that a false negative result occurs. The specific primer in the kit provided by the invention takes the Omp19 gene fragment as a target spot to realize the detection of the Brucella, and the kit further comprises specific immunomagnetic beads which can be used for enriching the Brucella pathogen, so that the high-efficiency detection of the Brucella can be realized, and an effective technical means is provided for the detection of the Brucella antigen. The test result shows that the lowest limit of the kit for detecting the brucella is 10⁵CFU/mL, for other fineThe bacteria detection is negative, no cross reaction occurs, the repeated variation coefficient in the group is 0.97-1.23%, the repeated variation coefficient between groups is 0.24-1.23%, and the repeatability is better; the method can also overcome the defects of long cycle time, complicated steps, time and labor waste and the like of the traditional detection method, greatly shortens the detection time, and meets the detection requirements of time and labor saving, rapidness and sensitivity.

Drawings

FIG. 1 shows the purification result of monoclonal IgG antibody against Brucella M5 antigen provided by the present invention; wherein, M: a protein Marker; 1: flowing through the liquid; 2: washing 1; 3: washing 2; 4: washing 3; 5: washing 4; 6: eluting 1; 7: eluting 2; 8: eluting 3;

FIG. 2 shows the result of detecting the specificity of immunomagnetic beads by qPCR method according to the present invention; wherein, S1: m5 vaccine liquid; s2: a19 vaccine bacterial liquid; s3: s2 vaccine liquid; s4: e.coli; s5: yersinia enterocolitica; s6: staphylococcus aureus bacteria; s7: salmonella liquid;

FIG. 3 shows the result of detecting the sensitivity of immunomagnetic beads by qPCR method according to the present invention; wherein, the concentration of M5 bacterial liquid is 1 × 10 respectively⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、10CFU/mL。

Detailed Description

The invention provides an immunomagnetic bead kit for detecting Brucella, which comprises: immunomagnetic beads, qPCR reaction mixed liquor, upstream primers, downstream primers and probes for detecting Brucella; the immunomagnetic beads are conjugates of mouse brucella resistance M5 antigen monoclonal IgG antibodies and the magnetic beads; the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, and the probe is a substance of which the 5 'end of the nucleotide sequence is marked with FAM and the 3' end is marked with BHQ1, and is shown as SEQ ID NO. 3. The invention preferably takes an Omp19 gene fragment (shown in SEQ ID NO: 4) as a target point, the selection of the Omp19 gene can ensure the reliability and stability of detection, and the primer pair for detecting the Brucella antigen is designed and synthesized. In the present invention, the nucleotide sequence of the upstream primer (Omp19F) is CCCGGTGAACTGGCTAATC (shown in SEQ ID NO: 1); the downstream primer (Omp19R) is TCGTAAAGGACGAGTTGCTTG (shown in SEQ ID NO: 2), and the Probe (Probe) is: FAM-CCTCCTGGGCCGTCAATGGCAAG-BHQ1, and the nucleotide sequence of the probe is CCTCCTGGGCCGTCAATGGCAAG (shown in SEQ ID NO: 3).

In the present invention, the kit further comprises a PBS buffer. In the present invention, the PBS buffer acts as a washing solution. In the present invention, the PBS buffer is preferably 10mM PBS buffer having a pH of 7.4.

In the invention, the immunomagnetic beads are stored in the form of immunomagnetic bead suspension, and the concentration of the immunomagnetic beads in the immunomagnetic bead suspension is 1 mg/mL; the solvent of the immunomagnetic bead suspension comprises PBS buffer. In the present invention, the PBS buffer is preferably 10mM PBS buffer having a pH of 7.4.

In the invention, the preparation method of the immunomagnetic beads comprises the following steps:

1) washing and resuspending the magnetic beads to obtain washed and resuspended magnetic beads;

2) mixing excessive mouse anti-brucella M5 antigen monoclonal IgG antibody with the washed and resuspended magnetic beads obtained in the step 1), and shaking for 15-60 min at 37 ℃ to obtain an immunomagnetic bead suspension;

3) and 2) carrying out magnetic adsorption on the immunomagnetic bead suspension obtained in the step 2), removing the supernatant, washing and resuspending to obtain the immunomagnetic beads.

The magnetic beads are washed and resuspended to obtain the washed and resuspended magnetic beads. The present invention preferably uses PBS buffer for washing and heavy suspension operation. In the present invention, the washing and resuspension operations are preferably performed in EP tubes. In the present invention, the magnetic beads are preferably magnetic nanospheres, and the magnetic nanospheres of the present invention are preferably purchased from wuhan jia source quantum dot technology development llc, cargo number: MNS-800). In the present invention, the stock solution of magnetic beads preferably contains 6mg of magnetic beads per 200. mu.L volume. In the invention, the time of magnetic adsorption is preferably 3-5min, and the magnetic adsorption is preferably carried out by adopting a magnetic frame. The source of the magnetic force frame is not particularly limited in the present invention, and a conventional commercially available magnetic force frame known to those skilled in the art may be used. In the present invention, the number of washing is preferably 2 to 4, and more preferably 3. In the present invention, the washing and resuspension are preferably performed using PBS buffer; the PBS buffer was 10mM PBS buffer at pH 7.4. When the present invention is performed for every 6mg of magnetic beads, it is preferable to perform resuspension using 600. mu.L PBS buffer.

After the washed and resuspended magnetic beads are obtained, mixing the excessive monoclonal IgG antibody of the mouse brucella resistance M5 antigen with the washed and resuspended magnetic beads, and shaking for 15-60 min at 37 ℃ to obtain an immunomagnetic bead suspension. In the present invention, the mass ratio of the monoclonal IgG antibody to the mouse anti-brucella M5 antigen to the magnetic beads is preferably 100 μ g/6mg or more. In the present invention, the time of the oscillation is preferably 30 min.

After the immunomagnetic bead suspension is obtained, the immunomagnetic bead suspension is subjected to magnetic adsorption, the supernatant is removed, and the immunomagnetic beads are obtained after washing and resuspension. In the present invention, the washing and resuspension are preferably performed using PBS buffer; the PBS buffer was 10mM PBS buffer at pH 7.4. In the present invention, the final concentration of the immunomagnetic beads is preferably 1 mg/mL.

In the invention, the use method of the immunomagnetic bead kit comprises the following steps:

(1) mixing immunomagnetic beads and a sample to be detected in a centrifuge tube, and carrying out oscillation reaction at 37 ℃ for 20-60 min;

(2) performing magnetic adsorption, removing the supernatant, washing and resuspending to obtain brucella-enriched immunomagnetic bead resuspension;

(3) processing the immune magnetic bead resuspension enriched with the Brucella in the step (2) by a thermal cracking method to obtain released Brucella DNA;

(4) mixing the Brucella DNA released in the step (3) with qPCR reaction mixed liquor, upstream primers, downstream primers and probes for detecting Brucella, and performing qPCR amplification;

(5) when the CT value is less than 28, the brucella is judged to be positive, and when the CT value is more than or equal to 28, the brucella is judged to be negative.

The invention mixes the immunomagnetic beads and a sample to be detected in a centrifuge tube, and vibrates and reacts for 20-60 min at 37 ℃. The enrichment of brucella (pathogen) is realized. In the present invention, the centrifuge tube is preferably a 1.5mL EP tube. In the present invention, the mixing volume ratio of the sample to be tested and the immunomagnetic beads is preferably 4 to 6, and more preferably 5, i.e., the amount of the immunomagnetic beads used for detecting each 100 μ L of the sample to be tested is preferably 20 μ L.

The invention enriches pathogeny, then carries out magnetic adsorption, discards supernatant, washes and re-suspends to obtain the brucella enriched immunomagnetic bead re-suspension. And completing washing of the pathogen-enriched immunomagnetic beads. In the present invention, the number of washing is preferably 2 to 4, and more preferably 3. In the present invention, the resuspension is preferably performed by adding PBS buffer solution with the volume 2-5 times that of the magnetic beads.

After obtaining the brucella enriched immunomagnetic bead resuspension, the invention adopts a thermal cracking method to process the brucella enriched immunomagnetic bead resuspension to obtain the released brucella DNA. In the present invention, the reaction procedure of the thermal cracking method in step 3) is: cracking at 95 deg.C for 10min, and ice-cooling for 3 min.

After the released Brucella DNA is obtained, the released Brucella DNA is mixed with qPCR reaction mixed liquor, upstream primers, downstream primers and probes for detecting Brucella, and qPCR amplification is carried out. In the present invention, the qPCR reaction mixture preferably comprises: (

Probe qPCR Supermix and RNase Free dH₂And O. Specifically, the qPCR amplification reaction system of the present invention preferably comprises, per 20 μ L:

probe qPCRSuperMix 10 mu L; 5 mu L of DNA template; RNase Free dH₂O2.6 mu L; upstream primer 0.8 μ L; 0.8 μ L of downstream primer and 0.8 μ L of probe primer. In the present invention, the reaction procedure of the qPCR amplification is preferably: 5min at 95 ℃; denaturation at 95 ℃ for 10s and renaturation at 60 ℃ for 30s for 40 cycles; storing at 4 ℃.

After amplification, the invention judges whether the sample contains the Brucella according to the CT value result of the qPCR reaction system. When the CT value is less than 28, the brucella is judged to be positive, and when the CT value is more than or equal to 28, the brucella is judged to be negative.

The immunomagnetic bead kit for detecting brucella antigen according to the present invention is further described in detail with reference to the following specific examples, and the technical solution of the present invention includes, but is not limited to, the following examples.