A kit for direct amplification detection of peste des petits ruminants virus based on single domain antibody 9-4-① immunomagnetic beads and its application

Abstract

The present invention discloses a kit for direct amplification detection of peste des petits ruminants virus based on single-domain antibody 9‑4‑① immunomagnetic beads and its application. The kit includes single-domain antibody 9‑4‑① immunomagnetic beads obtained by coupling the peste des petits ruminants virus single-domain antibody 9‑4‑① shown in SEQ ID NO.1 with streptavidin‑biotin and magnetic beads. The present invention uses gel-cutting purified single-domain antibody 9‑4‑① protein to couple with magnetic beads to obtain immunomagnetic beads. After the immunomagnetic beads are enriched with peste des petits ruminants virus, the genomic RNA does not need to be extracted and can be directly amplified. The proposal of the present invention overcomes the problems of low virus content, low detection rate, easy missed detection, etc.; as well as a series of difficulties such as time-consuming, costly, and labor-intensive when extracting genomic RNA, and polluting the environment during operation. The enrichment product is not only time-saving and resource-saving by directly amplifying, but also efficient, sensitive, specific, and stable. The proposal of the present invention provides an efficient technical means for the detection of peste des petits ruminants virus antigens.

Description

Technical Field

[0001] The invention relates to an immunomagnetic bead kit, in particular to a kit for detecting peste des petits ruminants virus by direct amplification based on single-domain antibody immunomagnetic beads and use thereof, and belongs to the field of biotechnology. Background Art

[0002] Peste des petits ruminants (PPR), also known as sheep plague, pseudorinderpest of small ruminants, and pneumoenteritis, is caused by peste des petits ruminants virus (PPRV), with fever, pneumonia, diarrhea, and dyspnea as the main clinical symptoms, and the morbidity and mortality rates can be as high as 100% and 90%, respectively. The use of vaccines has effectively controlled the large-scale outbreak of the epidemic, but in recent years, the occurrence of PPRV has almost never stopped. An important epidemiological survey of sheep diseases found that the persistent transmission of PPRV in wild blue sheep in the north is the key factor causing the current frequent sporadic or regional epidemics of PPRV (Lingxia Li, et al. Epidemic and Evolutionary Characteristics of Peste des Petits Ruminants Virus Infecting Procapraprzewalskii in Western China, Infection, Genetics and Evolution, 2019). This result brings new challenges to detection. The difficulty of continuous virus detection lies in the low virus content in the body and the complex interference factors, which can easily lead to missed detection, thus bringing certain difficulties to the accurate diagnosis and isolation of pathogens.

[0003] Single domain antibodies are the smallest antigen binding units of antibody molecules. They are composed of only one variable domain or an engineered constant domain that only assists in target binding. They do not contain the Fc segment of ordinary antibodies. Therefore, single domain antibodies can avoid the complement reaction caused by the Fc segment, so they are increasingly used in biological detection, diagnosis, pharmaceuticals and other fields. Immunomagnetic beads (IMB) is a new diagnostic technology based on antigen-antibody reaction and magnetic carrier technology. After a certain treatment, the antibody is bound to the magnetic beads. After the magnetic beads as antibody carriers bind to the antigen substance, an immune complex is formed. Under the action of the magnetic force of the magnet, this complex undergoes mechanical movement, separating the antigen that specifically binds to the antibody on the magnetic special from other substances. This technology has been applied to immunoassays, cell separation, protein purification and other aspects. Due to its unique characteristics of rapidity, high efficiency, low toxicity, high specificity and simple operation, it provides a new idea for antigen detection of latent infection.

[0004] The present invention combines the optimized single-domain antibody with the immunomagnetic bead technology, does not require complicated purification equipment and technology, and has no restrictions on the clarity of the sample. Only a simple magnetic adsorption step is required to conveniently detect low-trace levels of peste des petits ruminants virus, thereby establishing an immunoassay technology with fast detection speed, high specificity and sensitivity, and good repeatability. Summary of the invention

[0005] Aiming at a series of problems existing in the prior art, such as false negative results due to low virus content in the process of detecting PPR virus antigen, failure to meet the minimum requirements of PCR or Real-time-PCR detection, time-consuming and labor-intensive RNA extraction, and expensive purchase of RNA extraction kits, the present invention, on the one hand, utilizes a gel excision purification method to prepare high-purity single-domain antibody proteins, which has better protein purity and is more conducive to labeling than the common method, and the labeled immunomagnetic beads have stronger specificity in enriching PPR pathogens; on the other hand, the key link of genomic RNA extraction is eliminated, and the enriched products are directly amplified for real-time fluorescence quantitative detection, thereby saving time, manpower, material resources and financial resources, and improving economic benefits.

[0006] Specifically, in order to achieve the above purpose, the present invention adopts the following technical means:

[0007] The present invention discloses a kit for detecting peste des petits ruminants virus by direct amplification based on single-domain antibody 9-4-① immunomagnetic beads. The kit comprises single-domain antibody 9-4-① immunomagnetic beads obtained by antibody coupling of peste des petits ruminants virus single-domain antibody 9-4-① with magnetic beads through streptavidin-biotin (SA-Biotin), and the amino acid sequence of the peste des petits ruminants virus single-domain antibody 9-4-① is as shown in SEQ ID NO.1.

[0008] Among them, preferably, the peste des petits ruminants virus single-domain antibody 9-4-① is produced by a strain with a preservation number of CCTCC NO.M2019741 and a preservation name of Escherichia coli 9-4-① / PPRV (Escherichia coli 9-4-① / PPRV), and is obtained by a gel excision purification method.

[0009] Among them, preferably, the kit also includes an upstream primer, a downstream primer and a probe for direct amplification detection of peste des petits ruminants virus, and the sequences of the upstream primer, the downstream primer and the probe are respectively:

[0010] Upstream primer: 5′-GAGGGGAGTAGAGATGAAGACGAG-3′;

[0011] Downstream primer: 5′-ATAATTGGAAAGGCGGGCTAAGAC-3′;

[0012] Probe: 5′-GAGCTTGCAAGTTTCCTAATGGATCGG-3′.

[0013] Preferably, the kit also includes a direct amplification reaction solution.

[0014] Wherein, preferably, when the kit is used to detect peste des petits ruminants virus antigen, the following steps are followed:

[0015] 1) Virus enrichment: Take 10 μl of single-domain antibody 9-4-① immunomagnetic beads in a 1.5 mL EP tube, add 100 μl of the supernatant of the sample to be tested after centrifugation, shake and mix, place in a 4°C refrigerator, and enrich for 12 hours or overnight;

[0016] 2) Washing of magnetic beads: After the virus enrichment is completed, place the EP tube on a magnetic stand, discard the supernatant, wash three times with 1 ml of pH 7.6, 0.05 M PBS buffer, and finally resuspend with 50 μl of PBS buffer to obtain a suspension as the virus enrichment product;

[0017] 3) Establish a 50 μl direct amplification reaction system: 40 μl direct amplification reaction solution, 5 μl upstream and downstream primers and probe mixture, 5 μl suspension; amplification program: 50°C for 15 min; 95°C for 10 min; 95°C for 10 s, 60°C for 30 s, 72°C for 30 s, 5 cycles of pre-amplification; 95°C for 10 s, 51°C for 30 s, a total of 40 cycles and collect fluorescence;

[0018] The sequences of the primers and probes are:

[0019] Upstream primer: 5′-GAGGGGAGTAGAGATGAAGACGAG-3′;

[0020] Downstream primer: 5′-ATAATTGGAAAGGCGGGCTAAGAC-3′;

[0021] Probe: 5′-GAGCTTGCAAGTTTCCTAATGGATCGG-3′.

[0022] Preferably, the concentrations of the upstream and downstream primers and probes are all 10 μM.

[0023] Furthermore, the present invention also proposes the use of the kit in preparing a reagent for detecting peste des petits ruminants virus antigen.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The invention overcomes a series of problems such as low virus content during the detection of PPR virus antigen, which cannot meet the minimum requirements of RT-PCR detection and result in false negative results; time-consuming and labor-intensive RNA extraction; expensive RNA extraction kits; and low concentration and purity of single-domain antibodies after expression and purification. This application uses optimized single-domain antibodies with high expression levels and good reaction activity. Its immunomagnetic beads are more economical to use for pathogen enrichment. The kit obtained by directly amplifying the enriched product not only saves time and resources, but is also efficient, sensitive, specific, stable, and practical, providing an efficient technical means for the detection of PPR virus disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the SDS-PAGE result of gel-cleaved purified 9-4-① protein;

[0027] Left: Pre-stained protein marker; Right: 9-4-① protein target band;

[0028] Figure 2 This is the best binding result diagram of magnetic bead protein 9-4-①;

[0029] Figure 3 This is the result diagram of the enrichment sensitivity of magnetic bead protein 9-4-①;

[0030] Figure 4 This is a graph showing the specificity results of magnetic bead protein 9-4-① amplification.

[0031] Collection information:

[0032] Nomenclature: Escherichia coli 9-4-① / PPRV

[0033] Classification name: Escherichia coli 9-4-① / PPRV

[0034] Deposit number: CCTCC NO.M 2019741

[0035] Collection time: September 23, 2019

[0036] Preservation institution: China Center for Type Culture Collection (CCTCC for short).

[0037] Deposit address: Wuhan University, Wuhan, China DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are only used to illustrate the present invention and do not constitute any limitation to the scope of protection of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0039] Example 1 Screening of single-domain antibodies against peste des petits ruminants virus and construction of strains expressing the single-domain antibodies

[0040] 1.1 Materials Peste des Petits Ruminants virus single domain antibody library See patent: A camel-derived PPRV cDNA library and its preparation method and application (patent number ZL201810501728.0)

[0041] 1.2 Screening and construction of single-domain antibody 9-4-① against PPR virus

[0042] The antibody library in 1.1 was amplified using a two-step method. The VH-CH1-CH2 hinge region was amplified first, and the VH-CH1-CH2 band was recovered and directly amplified to obtain the VHH gene band (excluding the amplified part of the VHH-CH2 hinge region). The VHH gene was cloned and transformed, and several colony plaques were picked and placed in LB culture medium respectively. They were shaken and cultured within 12-14 hours, and small amounts of plasmids were extracted and sequenced for positive results. The sequences were analyzed and predicted one by one, and high-quality genes that met the single-domain antibody framework were selected and constructed into expression vectors pMal-c2X, and transferred into Escherichia coli to express their respective proteins in small amounts. The antibody effectiveness, i.e., reaction activity, was verified by antibody detection kits, classical virus neutralization experiments, and indirect immunofluorescence, and single-domain antibodies of peste des petits ruminants virus with high expression levels and good activity were screened out.

[0043] 1.3 Results

[0044] Several sequences were obtained by sequencing, and after comparison, analysis, small-scale expression, activity verification and other screening, a single-domain antibody 9-4-① against peste des petits ruminants virus was finally obtained. The amino acid sequence of the single-domain antibody 9-4-① protein is shown in SEQ ID NO. 1. The Escherichia coli expressing the single-domain antibody 9-4-① protein was named Escherichia coli 9-4-① / PPRV (Escherichia coli 9-4-① / PPRV) and preserved in the China Center for Type Culture Collection with the deposit number of CCTCC NO.M 2019741.

[0045] Example 2 Preparation of Peste des Petits Ruminants Virus Single Domain Antibody-Conjugated Immunomagnetic Beads (9-4-① Immunomagnetic Beads)

[0046] 2.1 Purification of single domain antibodies against PPR virus

[0047] Take the strain with the accession number of M 2019741, the amino acid sequence of the single-domain antibody 9-4-① protein expressed by the strain is as shown in SEQ ID NO.1, the expression vector is pMal-c2X, N-terminal-MBP solubility-aiding tag; C-terminal-HIS purification tag, draw 100 μl of the strain into 5 ml of LB medium containing ampicillin resistance, shake and culture, transfer to 1000 ml of LB medium containing ampicillin resistance, induce expression, collect the bacteria, ultrasonically lyse, centrifuge, collect the supernatant, purify with a nickel column affinity layer, and finally elute the protein bound to the column with 200 mM imidazole.

[0048] Under normal circumstances, the eluted protein is directly subjected to subsequent Elisa and other tests. However, it often contains foreign proteins, which affect the purity of the protein. The present application further performs gel cutting purification on the above protein, and the above protein is subjected to SDS-PAGE again, the target band is cut under clean conditions, placed in a high-pressure sterilized mortar, liquid nitrogen is added under safe conditions, ground, resuspended in 0.05M, pH7.6 PBS buffer, centrifuged at 4000rpm for 20min, the supernatant is collected, a small amount of liquid nitrogen is added to the precipitate, ground again, resuspended in PBS buffer, and the two resuspensions are concentrated using a 100,000 molecular weight cutoff (MW) ultrafiltration concentration system. The resulting product is a VHH 9-4-① / PPRV protein (8.5mg / ml) purified to a high purity and concentration. Figure 1 The conventional antibody titer is 1:320-1:640, which can be increased to at least 1:1280-1500 after purification.

[0049] 2.2 9-4-① Protein magnetic bead labeling

[0050] Add 1 ml of carboxyl magnetic beads to a centrifuge tube, place it on a magnetic rack, and discard the magnetic bead storage solution; wash it three times with pH 5.20.05M MES buffer, discard the buffer, add 1.6 times the amount of magnetic beads DEAC, and activate it on a rotating mixer for 30 minutes; add 20 μL of 9-4-① protein with a concentration of 8.5 mg / ml, vortex mix it at room temperature for 20 minutes, place the vortexed centrifuge tube on a magnetic rack for adsorption, discard the supernatant, add 1 ml of 1xPBS buffer to resuspend, wash, repeat 3 times, finally resuspend it with 4 ml of 1xPBS and add 1% BSA as a stabilizer for the marker, the resulting product is the 9-4-① protein magnetic bead marker, and use it to carry out the 9-4-① protein magnetic bead marker enrichment experiment of peste des petits ruminants virus attenuated seedlings.

[0051] Example 3 Optimal binding test of PPR virus single domain antibody coupled immunomagnetic beads (9-4-① immunomagnetic beads)

[0052] Buy attenuated vaccine powder of PPR virus and dilute it to about 10 3 TCID 50 / mL virus solution, each aspirate 100 μl, add 5 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl of peste des petits ruminants virus single domain antibody magnetic bead marker (prepared in Example 2) in sequence for enrichment, then wash with 1xPBS, and finally resuspend with 50 μl 1xPBS for real-time fluorescence amplification. The sequences of the upstream primer, downstream primer, and probe used are:

[0053] Upstream primer: 5′-GAGGGGAGTAGAGATGAAGACGAG-3′;

[0054] Downstream primer: 5′-ATAATTGGAAAGGCGGGCTAAGAC-3′;

[0055] Probe: 5′-GAGCTTGCAAGTTTCCTAATGGATCGG-3′.

[0056] A 50 μl direct amplification reaction system was established: 40 μl direct amplification reaction solution, 5 μl primer-probe mixture, and 5 μl resuspension solution; the amplification program was: 50°C for 15 min; 95°C for 10 min; 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s, for 5 cycles of pre-amplification; 95°C for 10 s, 51°C for 30 s, for a total of 40 cycles and fluorescence was collected to observe the enrichment of magnetic beads.

[0057] Results: From Figure 2 It can be seen that 10 μl of magnetic beads can be adsorbed economically and achieve optimal adsorption capacity.

[0058] Example 4 Optimal adsorption time of PPR virus single domain antibody-coupled immunomagnetic beads (9-4-① immunomagnetic beads)

[0059] In order to clarify the optimal time for the 9-4-① protein immunomagnetic beads to adsorb the PPR virus, take two 1.5 ml EP tubes, each of which is aspirated with 100 μl of the PPR virus attenuated vaccine dilution as in Example 3, add 10 μl of magnetic beads, place one at 4°C overnight and the other at 37°C for 2 hours, and perform quantitative amplification as in Example 3.

[0060] Results: The CT value after enrichment at 4℃ overnight was 16.28, and the CT value after enrichment at 37℃ for 2h was 19.01, which fully demonstrated that 4℃ overnight was the optimal reaction time for enrichment of PPR virus by 9-4-① protein immunomagnetic beads.

[0061] Example 5 Sensitivity of PPR virus single domain antibody coupled immunomagnetic beads (9-4-① immunomagnetic beads)

[0062] Take 1 mL of the attenuated vaccine solution of PPR virus in Example 3 (CT value is 16.12), and dilute it tenfold with 1xPBS to obtain 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Take 100 μl of the virus dilution and enrich with 10 μl of 9-4-① immunomagnetic beads at 4°C overnight, and test the sensitivity of 9-4-① immunomagnetic beads the next day.

[0063] According to the results Figure 3 According to the fluorescent quantitative PCR judgment standard, a CT value greater than 35 needs to be re-measured. Therefore, it can be seen from Table 1 that after enrichment, 10 -4 The diluted virus solution without magnetic bead enrichment can only detect 10 -3 Diluted virus solution. In addition, from the overall test results, the virus copy number content measured after enrichment is generally higher than that before enrichment. Explanation 9-4-① After immunomagnetic bead enrichment, the detection rate is significantly increased, and the sensitivity is increased by at least 10 times.

[0064] Example 6 Specificity of PPR virus single domain antibody coupled immunomagnetic beads (9-4-① immunomagnetic beads)

[0065] Take the immunomagnetic beads in Example 2, and follow the same steps as in Example 3 to enrich sheep pox, sheep ulcer and peste des petits ruminants attenuated vaccines for specific detection.

[0066] The results are as follows Figure 4 , the positive control and PPR samples showed peaks, while the negative control, sheeppox and sheep sore attenuated vaccines did not show peaks, indicating that the 9-4-① immunomagnetic beads are specific to PPR virus and have strong specificity.

[0067] Example 7 Kit for detecting peste des petits ruminants virus based on direct amplification of immunomagnetic beads coupled to peste des petits ruminants virus single domain antibody

[0068] The kit includes immunomagnetic beads coupled to the peste des petits ruminants virus single domain antibody 9-4-① prepared in Example 2, a direct amplification reaction solution, and a primer-probe mixture. The sequences of the upstream primer, downstream primer, and probe used are:

[0069] Upstream primer: 5′-GAGGGGAGTAGAGATGAAGACGAG-3′;

[0070] Downstream primer: 5′-ATAATTGGAAAGGCGGGCTAAGAC-3′;

[0071] Probe: 5′-GAGCTTGCAAGTTTCCTAATGGATCGG-3′.

[0072] A 50 μl direct amplification reaction system was established: 40 μl direct amplification reaction solution, 5 μl primer-probe mixture, and 5 μl resuspension solution; the amplification reaction conditions were: 50°C for 15 min; 95°C for 10 min; 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s, for 5 cycles of pre-amplification; 95°C for 10 s, 51°C for 30 s, for a total of 40 cycles and fluorescence collection. Sequence Listing <110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences <120> A kit for direct amplification detection of peste des petits ruminants virus based on single domain antibody 9-4-① immunomagnetic beads and its application <141> 2022-05-12 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 122 <212> PRT <213> peste des petits ruminants virus antibody <400> 1 His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Val 20 25 30 His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gly Trp Val 35 40 45 Ser Ser Ile Ser Asn Gly Gly Val Ser Thr Met Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Trp 65 70 75 80 Leu Gln Leu Asn Ser Leu Gln Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Lys Leu Gly Trp Ser Pro Lys Glu Ala Leu Asp Pro Ser Pro Ser Arg 100 105 110 Gly Arg Gly Thr Gln Val Thr Val Ser Ser 115 120

Claims

1. A kit for detecting peste des petits ruminants virus by direct amplification of single domain antibody 9-4-① immunomagnetic beads, characterized in that: The kit comprises single-domain antibody 9-4-① obtained by coupling the single-domain antibody 9-4-① of peste des petits ruminants virus with magnetic beads through streptavidin-biotin (SA-Biotin), and upstream primers, downstream primers and probes for direct amplification detection of peste des petits ruminants virus; The amino acid sequence of the peste des petits ruminants virus single domain antibody 9-4-① is shown in SEQ ID NO.1; The sequences of the upstream primer, downstream primer and probe are respectively: Upstream primer: 5′-GAGGGGAGTAGAGATGAAGACGAG-3′; Downstream primer: 5′-ATAATTGGAAAGGCGGGCTAAGAC-3′; Probe: 5′-GAGCTTGCAAGTTTCCTAATGGATCGG-3′.

2. The kit according to claim 1, characterized in that The peste des petits ruminants virus single domain antibody 9-4-① is produced by a strain with a deposit number of CCTCC NO.M 2019741 and a deposit name of Escherichia coli 9-4-① / PPRV (Escherichia coli9-4-① / PPRV), and is obtained by a gel excision purification method.

3. The kit according to claim 1, characterized in that The kit also includes a direct amplification reaction solution.

4. The kit according to claim 1, characterized in that When using the kit for detecting PPR virus antigen, follow the steps below: 1) Virus enrichment: Take 10 μl of single-domain antibody 9-4-① immunomagnetic beads in a 1.5 mL EP tube, add 100 μl of the supernatant of the sample to be tested after centrifugation, shake and mix, place in a 4°C refrigerator, and enrich for 12 hours or overnight; 2) Washing of magnetic beads: After the virus enrichment is completed, place the EP tube on a magnetic stand, discard the supernatant, wash three times with 1 ml of pH 7.6, 0.05 M PBS buffer, and finally resuspend with 50 μl of pH 7.6, 0.05 M PBS buffer to obtain a suspension as the virus enrichment product; 3) Establish a 50 μl direct amplification reaction system: 40 μl direct amplification reaction solution, 5 μl upstream and downstream primers and probe mixture, 5 μl suspension; amplification program: 50°C for 15 min; 95°C for 10 min; 95°C for 10 s, 60°C for 30 s, 72°C for 30 s, 5 cycles of pre-amplification; 95°C for 10 s, 51°C for 30 s, a total of 40 cycles and collect fluorescence; The sequences of the primers and probes are: Upstream primer: 5′-GAGGGGAGTAGAGATGAAGACGAG-3′; Downstream primer: 5′-ATAATTGGAAAGGCGGGCTAAGAC-3′; Probe: 5′-GAGCTTGCAAGTTTCCTAATGGATCGG-3′.

5. The kit according to claim 4, characterized in that The concentrations of the upstream and downstream primers and probes are all 10 μM.

6. Use of the kit according to any one of claims 1 to 5 in preparing a reagent for detecting peste des petits ruminants virus antigen.

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