Preparation method and application of crab cactus X virus CP protein polyclonal antibody
By cloning and expressing the CP gene of Crab Caryn X virus, polyclonal antibodies were prepared and Dot-blot-ELISA detection method was established, which solved the problems of false positives and complex and expensive detection of Dragon Fruit virus in the prior art, and achieved rapid , accurate field testing meets the needs of large-scale census.
Patent Information
- Application Number
- CN202411186887.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-28
- Publication Date
- 2024-12-06
AI Technical Summary
The prior art is prone to false positives when detecting dragon fruit virus, and the detection methods are complex and expensive, making it difficult to meet the needs of rapid and large-scale testing in the field.
Cloning the CP gene encoded by Crab Caryn X virus (ZyVX), constructing a prokaryotic expression vector, prokaryotic expression and purifying to obtain highly immunogenic ZyVX CP protein, preparing specific polyclonal antibodies, and establishing rapid Dot-blot-ELISA detection method.
It improves the sensitivity and accuracy of the detection, provides technical solutions for field prevention and control and research, and can quickly and accurately detect ZyVX infected samples of dragon fruit, with an accuracy rate of 88.9%.
Smart Images
Figure CN119080920A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of polyclonal antibodies, and in particular relates to a preparation method and application of a polyclonal antibody against CP protein of Schlumbergera X virus. Background Art
[0002] Pitaya (Hylocereus spp.) is a perennial plant of the genus Hylocereus in the family Cactaceae. In Guizhou, pitaya is mainly grown in the low-altitude, heat-rich river valleys in the south. The hot and rainy climate makes pitaya virus disease common in these areas, which has become an important factor restricting the development of the pitaya industry. The preparation of polyclonal antibodies to pitaya virus and the establishment of rapid detection technology can provide a scientific basis for the prediction and early prevention and control of the virus disease in production, and can also provide auxiliary technical means for the study of viruses. The RNA viruses that infect pitaya are mainly viruses of the genus Potexvirus of the family Alphaflexiviridae, including Zygocactus virus X (ZyVX), Pitaya virus X (PiVX), Schlumbergera virus X (SchVX) and Cactus virus X (CVX). Recently, it was reported that Pitayabadnavirus 1 (PiBV1) of the Badnavirus genus (Baduvirus) of the Caulimoviridae family also infects pitaya in Guizhou. [9]. ZyVX is a monopartite positive-sense single-stranded RNA virus containing 5 open reading frames (ORFs). ORF1 encodes RNA-dependent RNA polymerase (RdRP), ORF2-4 encode TGB1-3 proteins (Trige gene block 1-3), and ORF5 encodes coat protein (CP). Immunological methods are one of the most widely used methods for plant virus detection. They have the advantages of low cost, good repeatability, simple operation, and high sensitivity. Among them, enzyme-linked immunosorbent assay (ELISA) is particularly suitable for large-scale sample screening. However, due to the influence of factors such as virus variation and different hosts, this serological detection method is prone to false positives. In order to make up for the shortcomings of serological detection methods, it is a current trend to combine this method with other methods to improve the sensitivity and accuracy of detection. Dot-blot-ELISA combines the advantages of ELISA and Western blot. It is more convenient and faster to operate than ELISA, requires less antigen, and has a more sensitive and intuitive reaction. Summary of the invention
[0003] The technical problem to be solved by the present invention is to provide a method for preparing polyclonal antibodies to the CP protein of Zygcactus cactus X virus and its application in view of the deficiencies of the above-mentioned prior art. The method clones the CP gene encoded by ZyVX, constructs a prokaryotic expression vector, expresses and purifies the ZyVX CP protein with high immunogenicity, prepares specific polyclonal antibodies to ZyVX CP, and establishes a rapid Dot-blot-ELISA detection method for ZyVX, thereby providing a technical solution for field prevention and control and research of ZyVX.
[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing polyclonal antibodies against the CP protein of Schlumbergera X virus, the method comprising:
[0005] S1. RNA extraction and RT-PCR:
[0006] Extract total RNA from pitaya stems infected with ZyVX to obtain total RNA, use the obtained total RNA as a template, use ZyVX-CP-F and ZyVX-CP-R as primers to perform RT-PCR amplification, and recover and purify the PCR product after detection by 1% agarose gel electrophoresis to obtain a ZyVX CP gene fragment; the nucleotide sequence of the ZyVX CP gene fragment is shown in SEQ ID NO: 1; the nucleotide sequence of the ZyVX-CP-R is shown in SEQ ID NO: 2; the nucleotide sequence of the ZyVX CP gene fragment is shown in SEQ ID NO: 3;
[0007] S2. Construction of recombinant prokaryotic expression vector:
[0008] The ZyVX CP gene fragment obtained in S1 and pET28a were double-digested with Ndel and SacⅠ, respectively, and then connected to obtain a recombinant expression vector pET28a-CP, and the recombinant expression vector pET28a-CP was transformed into E. coli DH5α, and PCR colony identification was performed. After the PCR product was sequenced, the recombinant plasmid was extracted and transformed into E. coli Rosetta to obtain colonies carrying the recombinant plasmid;
[0009] S3, ZyVX CP protein prokaryotic expression and protein purification:
[0010] A single colony with the recombinant plasmid obtained in S3 was picked up and placed in LB liquid culture medium, placed in a shaker, and shaken at 37°C. When the bacterial solution became turbid and the OD value was measured to be 0.5, 1mM IPTG was added to the bacterial solution, placed in a shaker, and shaken at 37°C for 8h; the sample was centrifuged at 12000rpm for 3min, and the precipitate was retained; the lysis buffer was added, the centrifuge tube was shaken to resuspend the bacterial cells, and then 5× Loading Buffer was added, placed in a metal bath, allowed to stand at 98°C for 10min, and centrifuged at 10000rpm for 10min. The bacterial cells in 300mL of the induced expression solution were collected, and the bacterial cells were washed with 10mL of 0.01M Resuspend in PBS solution; ultrasonicate at 100W for 16min in an ice-water bath; centrifuge at 4°C, 12000rpm for 10min to collect the precipitate and supernatant respectively, retain 20μL of the supernatant for SDS-PAGE electrophoresis detection; resuspend and disperse the precipitate with 10mL of 8M urea solution without imidazole, ultrasonicate at 100W for 10min in an ice-water bath, retain 20μL of the sample for electrophoresis detection, purify the His-tag protein with Ni-NTA Resin to obtain a purified CP fusion protein, and then dialyze the purified CP fusion protein with dialysis buffers containing 6M, 4M, 3M, 2M, 1M, and 0M urea, respectively, to obtain a purified CP protein;
[0011] S4. Preparation of ZyVX CP antiserum:
[0012] The purified CP protein obtained in S3 was used as an antigen to immunize rabbits 4 times in total, and the adjuvant and the antigen were extracted at a volume ratio of 1:1. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second to fourth immunizations. The rabbits were injected subcutaneously at five points, with 0.2 mL at each point. The second immunization was performed on the 14th day after the first immunization, and the third immunization was performed 7 days after the second immunization. A small sample of serum was collected from the middle ear artery of the rabbit on the 7th day after the third immunization for detection. After the test was qualified, an additional immunization was performed 7 days later. Whole blood was collected 7 days after the additional immunization, and the supernatant was collected by centrifugation. The antibody was purified by dialysis to obtain a polyclonal antibody to the CP protein of Schlumbergera X virus.
[0013] The present invention also provides the use of the polyclonal antibody against the CP protein of Schlumbergera X virus prepared by the above-mentioned preparation method, wherein the polyclonal antibody against the CP protein of Schlumbergera X virus is used to detect pitaya ZyVX-infected samples.
[0014] Preferably, the method for detecting ZyVX-infected samples of pitaya is Dot-blot-ELISA detection of ZyVX, which is:
[0015] After weighing the pitaya stem, add 0.01mol / L PBS at a weight-to-volume ratio of 1:10 (g / ml) and grind it, centrifuge at 8000rpm for 3min; take 2μL of the supernatant and apply it to the NC membrane, let it stand for 10min; block the NC membrane in PBST with a final concentration of 5% skim milk powder for 30min, wash the membrane, add a primary anti-Zygocactus X virus CP protein polyclonal antibody at a ratio of 1:5000 in 1×PBST containing 5% skim milk powder, put it into the NC membrane, incubate it at room temperature for 2h, and wash the membrane; put the NC membrane into a goat anti-rabbit-HRP secondary antibody at a ratio of 1:10000 in 1×PBST and incubate it at room temperature for 1h, wash the membrane; add DAB colorimetric solution and observe the color development; the result is a qualitative result, color development is positive, and no color development is negative.
[0016] Compared with the prior art, the present invention has the following advantages:
[0017] 1. The present invention cloned the CP gene encoded by ZyVX, constructed a prokaryotic expression vector, prokaryotically expressed and purified the highly immunogenic ZyVX CP protein, prepared specific polyclonal antibodies to ZyVX CP, and established a rapid Dot-blot-ELISA detection method for ZyVX, providing a technical solution for field prevention and control and research of ZyVX.
[0018] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1This is a PCR verification diagram of the construction of the prokaryotic expression vector of the ZyVX CP gene of the present invention. (In the figure, M, DL2000 marker; 1, PCR amplification product of the prokaryotic expression vector of the ZyVX CP gene; 2, negative control.)
[0020] Figure 2 This is an SDS-PAGE analysis diagram of the purified product of the induced expression of the ZyVX CP protein of the present invention. (M in the figure: Protein Marker; 1, total protein; 2, Ni-NTA purified effluent; 3-4, purified ZyVX CP recombinant protein.)
[0021] Figure 3 This is a sensitivity detection diagram of the ZyVX CP protein polyclonal antibody of the present invention.
[0022] Figure 4 Specific detection of the ZyVX CP polyclonal antibody of the present invention. (In the figure, 1-9, 11-19, samples to be tested; 10, positive control; 20, negative control.) DETAILED DESCRIPTION
[0023] Example 1
[0024] Test plants: ZyVX-infected pitaya stems collected from Luodian and preserved in this laboratory.
[0025] Reagents: BCA Protein Assay Kit (Thermo Fisher Scientific), NC membrane (abcam), D2500 Gel Extraction Kit (OMEGA), Goldenstar TM RT6cDNA Synthesis Kit (Beijing Qingke Biotechnology Co., Ltd.), PCR Master Mix (Shanghai Yisheng Biotechnology Co., Ltd.), DAB horseradish peroxide colorimetric kit (Beyotime Biotechnology Co., Ltd.), prokaryotic expression vector pET28a (Beijing Qingke Biotechnology Co., Ltd.), Escherichia coli DH5α (Beijing Qingke Biotechnology Co., Ltd.), Escherichia coli Rosetta (Beijing Qingke Biotechnology Co., Ltd.).
[0026] The method for preparing polyclonal antibodies against the CP protein of Schlumbergera X virus of this embodiment is as follows:
[0027] S1. RNA extraction and RT-PCR:
[0028] Extract total RNA from pitaya stems to obtain total RNA, use biological software to take the ZyVX virus CP gene sequence downloaded from NCBI as a template sequence for detection, design primers, use the obtained total RNA as a template, use ZyVX-CP-F and ZyVX-CP-R as primers, use a one-step RT-PCR kit to perform RT-PCR amplification, and after the PCR product is detected by 1% agarose gel electrophoresis, use a DNA gel purification recovery kit to recover and purify, and obtain a ZyVX CP gene fragment; the nucleotide sequence of the ZyVX CP gene fragment is shown in SEQ ID NO: 1; the nucleotide sequence of the ZyVX-CP-R is shown in SEQ ID NO: 2; the nucleotide sequence of the ZyVXCP gene fragment is shown in SEQ ID NO: 3;
[0029] The total RNA from pitaya stem infected with ZyVX (Zygocactus virus X) was used as a template to amplify the ZyVX CP gene by RT-PCR. The results of 1% agarose gel electrophoresis showed that a specific band of about 700 bp was obtained by PCR amplification ( Figure 1 ), and the sequencing results after purification and recovery showed that the band size was 681 bp, and the homology with the ZyVX CP gene (Genbank accession number: KM288845) was 98.83%;
[0030] S2. Construction of recombinant prokaryotic expression vector:
[0031] The ZyVX CP gene fragment obtained in S1 and pET28a were double-digested with Ndel and SacⅠ, respectively, and then connected to obtain a recombinant expression vector pET28a-CP, and the recombinant expression vector pET28a-CP was transformed into E. coli DH5α, and PCR colony identification was performed using the T7 primer provided in the prokaryotic expression vector pET28a kit. After the PCR product was sequenced, the recombinant plasmid was extracted and transformed into E. coli Rosetta to obtain colonies carrying the recombinant plasmid;
[0032] The prokaryotic expression vector pET28a kit was purchased from Beijing Qingke Biotechnology Co., Ltd.
[0033] S3, ZyVX CP protein prokaryotic expression and protein purification:
[0034] A single colony with the recombinant plasmid obtained in S3 was picked and placed in LB liquid culture medium, placed in a shaker, and shaken at 37°C. When the bacterial solution became turbid and the OD value was measured to be 0.5, 1mM IPTG was added to the bacterial solution, placed in a shaker, and shaken at 37°C for 8h; the sample was centrifuged at 12000rpm for 3min, and the precipitate was retained; the lysis solution was added, the centrifuge tube was shaken to resuspend the bacterial cells, and then 5× Loading Buffer was added, placed in a metal bath, and allowed to stand at 98°C for 10min, and the results were tested by SDS-PAGE; the bacterial cells in 300mL of the induced expression solution were collected by centrifugation at 10000rpm for 10min, and the bacterial cells were washed with 10mL of 0.01M Resuspend in PBS solution; ultrasonicate at 100W for 16min in an ice-water bath; centrifuge at 4°C, 12000rpm for 10min to collect the precipitate and supernatant respectively, retain 20μL of the supernatant for SDS-PAGE electrophoresis detection; resuspend and disperse the precipitate with 10mL of 8M urea solution without imidazole, ultrasonicate at 100W for 10min in an ice-water bath, retain 20μL of the sample for electrophoresis detection, purify the His-tag protein with Ni-NTA Resin to obtain a purified CP fusion protein, and then dialyze the purified CP fusion protein with dialysis buffer containing 6M, 4M, 3M, 2M, 1M, and 0M urea, respectively, to obtain a purified CP protein; the concentration of the purified protein is determined using a BCA protein concentration determination kit;
[0035] After the ZyVX CP gene was induced to express, the CP protein was purified by Ni-NTA agarose affinity chromatography. SDS-PAGE electrophoresis showed that a soluble protein was expressed at about 25 kDa, which was basically the same as the amino acid molecular size of the fusion protein of the CP protein amino acid sequence plus the His-tag sequence ( Figure 2 );
[0036] S4. Preparation of ZyVX CP antiserum:
[0037] The purified CP protein obtained in S3 was used as an antigen to immunize rabbits 4 times in total, and the adjuvant and the antigen were extracted at a volume ratio of 1:1. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second to fourth immunizations. The rabbits were injected subcutaneously at five points, with 0.2 mL at each point. The second immunization was performed on the 14th day after the first immunization, and the third immunization was performed 7 days after the second immunization. A small sample of serum was collected from the middle ear artery of the rabbit on the 7th day after the third immunization for detection. After the test was qualified, an additional immunization (fourth immunization) was performed 7 days later. Whole blood was collected 7 days after the additional immunization, and the supernatant was collected by centrifugation. The antibody was purified by dialysis to obtain a polyclonal antibody to the CP protein of Schlumbergera X virus.
[0038] This embodiment also provides the use of the polyclonal antibody against the CP protein of Schlumbergera X virus prepared by the preparation method, wherein the polyclonal antibody against the CP protein of Schlumbergera X virus is used to detect pitaya ZyVX-infected samples.
[0039] (1) Indirect ELISA to detect antibody titer:
[0040] Dilute the ZyVX CP antigen to 1 μg / ml with 1×PBST, add 50 μl to each reaction well of the polystyrene plate, incubate at 4°C overnight, discard the solution in the wells, wash once with 1×PBST washing buffer at 200 μl per well. Add 60 μl of 1% BSA (prepared with PBST) to each well for blocking, and incubate at 37°C for 1 hour. Discard the blocking solution. Add 50 μl of the antibody diluted in a gradient from 250 to 1024000 times to the above blocked reaction wells; at the same time, set up the positive control well (positive serum) and the negative control well (BSA); incubate at 37°C for 1 hour, then discard the blocking solution, and wash twice with 1×PBST washing buffer at 200 μl per well. Freshly diluted secondary antibody (HRP labeled, 1:5000, diluted with 1% BSA) was added to the ELISA plate at 50 μl / well, incubated at 37°C for 45 min, then the blocking solution was discarded and washed 3 times with 1×PBST buffer at 200 μl per well; 100 μl of temporarily prepared TMB substrate solution was added to each reaction well and reacted at 37°C for 5 min; 50 μl of 2M sulfuric acid was added to each reaction well to terminate the reaction; the absorbance was measured at OD450 nm, and 2.5 times the absorbance of the negative control OD450nm was taken as the antibody titer.
[0041] When the ZyVX CP antigen was diluted to 1 μg / ml, the antibody titer was measured. The results showed that when the dilution concentration was 1:1024000, that is, when the antibody concentration was 13.3 ng / ml, the reading at OD450nm was still 2.5 times greater than the negative control (1% BSA), indicating that its titer could reach 1:1024000 ( Figure 3 ).
[0042] (2) Dot-blot-ELISA specificity determination:
[0043] After weighing the pitaya stem, add 0.01mol / L PBS at a weight-volume ratio of 1:10 (g / ml) and grind it. Centrifuge at 8000rpm for 3min; take 2μl of the supernatant and apply it to the NC membrane, let it stand for 10min; block the NC membrane in PBST with a final concentration of 5% skim milk powder for 30min, and wash the membrane. Add the primary antibody at a ratio of 1:5000 in 1×PBST containing 5% skim milk powder, put it in the NC membrane, incubate it at room temperature for 2h, and wash the membrane; put the NC membrane in the goat anti-rabbit-HRP secondary antibody at a ratio of 1:10000 in 1×PBST and incubate it at room temperature for 1h, wash the membrane; add DAB colorimetric solution and observe the color development. The result is a qualitative result, color development is positive, and no color development is negative.
[0044] Dot-blot-ELISA results are as follows Figure 4 , with ZyVX-infected pitaya as the positive control, CK+ showed obvious coloration, and with healthy pitaya as the negative control, CK- showed no coloration, and the tested samples could show color accurately. This indicates that the prepared ZyVX CP protein polyclonal antibody can specifically identify pitaya samples infected with ZyVX virus.
[0045] The antibody was used to detect 144 pitaya samples, which were collected from Bacao Village, Zhenning County. The variety was Guihonglong. Compared with the RT-PCR method, the accuracy rate reached 88.9%.
[0046] As a structural protein of the virus, CP can protect the genetic material inside the virus and also largely determine the virus strain type, infection, transmission medium identification, symptom manifestation, long-distance transport and host range and other viral biological characteristics and serological characteristics. The differences between plant virus strains are often related to the differentiation of CP. CP is also the most abundant viral protein in cells. In the immunological identification of viruses, CP antibodies are the most commonly used antibodies. In this study, the CP protein of ZyVX was expressed in prokaryotes and used to immunize animals to obtain polyclonal antibodies to ZyVX CP.
[0047] At present, the detection and identification of pitaya virus mainly rely on high-throughput sequencing and molecular biology detection method reverse transcription-polymerase chain reaction (RT-PCR). Compared with the molecular biology detection method, the advantage of high-throughput sequencing is that it does not require a prior understanding of the genomic characteristics of the virus, which is conducive to the discovery of new viruses. The disadvantage is that the cost is high, the amount of data is large, and the analysis is not easy. The molecular biology method RT-PCR has the characteristics of high sensitivity and good specificity, but the disadvantage is that the reagents are expensive, the steps are complicated, and the detection time is long. The present invention has developed a serological detection method for ZyVX, which has simple steps, large throughput, and fast speed, but after all, polyclonal antibodies are used, and the specificity is poorer than monoclonal antibodies, and there is a possibility of false positives. If used in scientific research, it should be used in combination with other detection methods.
[0048] ZyVX infects pitaya, affecting its quality and yield, seriously affecting the planting income of pitaya. In addition, the virus can also infect pitaya with multiple viruses, including CVX, PiVX and SchVX, and the symptoms are more serious than those of a single virus infection. The Dot-blot-ELISA detection method for ZyVX established in this study can be used for the rapid detection of ZyVX and can be used for the detection of field crop samples, thus providing a scientific method for large-scale field ZyVX surveys.
[0049] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.
Claims
1. A method for preparing polyclonal antibodies against Schlumbergera X virus CP protein, characterized in that: The method is: S1. RNA extraction and RT-PCR: Extract total RNA from pitaya stems infected with ZyVX to obtain total RNA, use the obtained total RNA as a template, use ZyVX-CP-F and ZyVX-CP-R as primers to perform RT-PCR amplification, and recover and purify the PCR product after detection by 1% agarose gel electrophoresis to obtain a ZyVX CP gene fragment; the nucleotide sequence of the ZyVX CP gene fragment is shown in SEQ ID NO: 1; the nucleotide sequence of the ZyVX-CP-R is shown in SEQ ID NO: 2; the nucleotide sequence of the ZyVX CP gene fragment is shown in SEQ ID NO: 3; S2. Construction of recombinant prokaryotic expression vector: The ZyVX CP gene fragment obtained in S1 and pET28a were double-digested with Ndel and SacⅠ, respectively, and then connected to obtain a recombinant expression vector pET28a-CP, and the recombinant expression vector pET28a-CP was transformed into E. coli DH5α, and PCR colony identification was performed. After the PCR product was sequenced, the recombinant plasmid was extracted and transformed into E. coli Rosetta to obtain colonies carrying the recombinant plasmid; S3, ZyVX CP protein prokaryotic expression and protein purification: Pick a single colony from the colonies with the recombinant plasmid obtained in S3 and put it into LB liquid culture medium, put it into a shaker, and shake at 37°C. When the bacterial solution is turbid and the OD value is 0.5, add 1mM IPTG to the bacterial solution, put it into a shaker, and shake at 37°C for 8h; centrifuge the sample at 12000rpm for 3min, and keep the precipitate; add lysis buffer, shake the centrifuge tube to resuspend the bacterial cell, and then add 5× Loading Buffer, put it in a metal bath, let it stand at 98°C for 10min, centrifuge it at 10000rpm for 10min, collect the bacteria in 300mL of bacterial liquid after induced expression, and resuspend the bacteria with 10mL of 0.01M PBS solution with a pH of 7.4; ultrasonicate it at 100W for 16min under ice-water bath; centrifuge it at 4°C and 12000rpm for 10min to collect the precipitate and supernatant respectively, and keep 20μL of the supernatant for SDS-PAGE electrophoresis detection; resuspend the dispersed precipitate with 10mL of 8M urea solution without imidazole, ultrasonicate it at 100W for 10min under ice-water bath, keep 20μL of the supernatant for electrophoresis detection, purify the His-tag protein with Ni-NTA Resin to obtain the purified CP fusion protein, and then dialyze the purified CP fusion protein with dialysis buffer containing 6M, 4M, 3M, 2M, 1M, and 0M urea, respectively, to obtain the purified CP protein; S4. Preparation of ZyVX CP antiserum: The purified CP protein obtained in S3 was used as an antigen to immunize rabbits 4 times in total, and the adjuvant and the antigen were extracted at a volume ratio of 1:
1. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second to fourth immunizations. The rabbits were injected subcutaneously at five points, with 0.2 mL at each point. The second immunization was performed on the 14th day after the first immunization, and the third immunization was performed 7 days after the second immunization. A small sample of serum was collected from the middle ear artery of the rabbit on the 7th day after the third immunization for detection. After the test was qualified, an additional immunization was performed 7 days later. Whole blood was collected 7 days after the additional immunization, and the supernatant was collected by centrifugation. The antibody was purified by dialysis to obtain a polyclonal antibody to the CP protein of Schlumbergera X virus.
2. An application of a polyclonal antibody against Schlumbergera X virus CP protein prepared by the preparation method according to claim 1, characterized in that: The polyclonal antibody against the CP protein of Schlumbergera X virus is used to detect pitaya ZyVX-infected samples.
3. The use according to claim 2, characterized in that: The method for detecting pitaya ZyVX infected samples is Dot-blot-ELISA detection of ZyVX, and the method is: After weighing the pitaya stem, add 0.01mol / L PBS at a weight-to-volume ratio of 1:10 (g / ml) and grind it, centrifuge at 8000rpm for 3min; take 2μL of the supernatant and apply it to the NC membrane, let it stand for 10min; block the NC membrane in PBST with a final concentration of 5% skim milk powder for 30min, wash the membrane, add a primary anti-Zygocactus X virus CP protein polyclonal antibody at a ratio of 1:5000 in 1×PBST containing 5% skim milk powder, put it into the NC membrane, incubate it at room temperature for 2h, and wash the membrane; put the NC membrane into a goat anti-rabbit-HRP secondary antibody at a ratio of 1:10000 in 1×PBST and incubate it at room temperature for 1h, wash the membrane; add DAB colorimetric solution and observe the color development; the result is a qualitative result, color development is positive, and no color development is negative.