Secreted monoclonal antibody hybridoma cell strain against rice bacterial leaf blight and monoclonal antibody application thereof

Abstract

The application discloses a hybridoma cell strain secreting a monoclonal antibody against Xanthomonas oryzae pv. oryzae (Xoo) and application of the monoclonal antibody. A BALB / c mouse is immunized by using a cultured Xoo Y2 strain as an antigen, and a hybridoma cell strain 19C9 capable of secreting the monoclonal antibody against the Xoo is obtained through a hybridoma technology, and the strain is preserved with a preservation number of CGMCC No. 46004. The indirect ELISA titer of the monoclonal antibody secreted by the cell strain reaches 10 ‑7 , the antibody type and subclass are IgG1 and kappa light chain, the monoclonal antibody has specific immunoreactions with different strains or isolates of the Xoo, does not react with other bacteria such as rice bacterial leaf streak bacteria and rice panicle blast bacteria, and does not have an immunoreaction with healthy rice plant tissues. Dot-ELISA and immunocolloidal gold test strip two serological methods for detecting the Xoo are established by using the 19C9 monoclonal antibody. The hybridoma cell strain, the preparation of the monoclonal antibody secreted by the hybridoma cell strain and the establishment of the serological detection method provide detection reagents and technical support for detection and diagnosis, epidemiological investigation and scientific prevention and control of the Xoo.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a hybridoma cell strain secreting monoclonal antibody against Xanthomonas oryzae pv. oryzae and application of the monoclonal antibody. BACKGROUND

[0002] Rice is one of the most important food crops for human beings. However, bacterial diseases seriously harm rice and cause huge yield loss every year. Among them, bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterium, is one of the three major diseases of rice and seriously restricts the yield of rice. After rice is infected with BLB, the yield is usually reduced by 10%-30%, and in severe cases, the yield is reduced by more than 50% or even no yield. BLB has a wide range of occurrence, and it has been found in all continents except Antarctica. Among them, China, Japan and India in Asia are most seriously affected by the disease.

[0003] BLB of rice can occur at all growth stages of rice and shows leaf blight symptoms. The stomata of rice leaves are located on the upper edge of the leaves, so the disease usually spreads from the leaf edge near the stomata. As the disease progresses, the lesion changes from water-stained color to yellow, the area increases and gradually develops into an elongated irregular lesion, the edge is wavy, and the lesion is composed of small yellow spherical substances. When the air humidity is high, the granular colonies on the diseased leaves will become soft mud-like pus. With the passage of time, the lesion area may cover the entire leaf and make the leaf appear white or gray. The pathogen Xoo usually enters the rice leaf through the stomata at the leaf tip and edge. The pathogen on the leaf surface may be suspended in the water droplets on the leaf surface at night along with the formation of dew and enter the plant by flagellum swimming, or be passively absorbed into the leaf vascular tissue in the morning along with the absorption of water droplets by the leaf, and then multiply in the intercellular space of the vascular tissue and spread to the plant through the xylem. The pathogen can also enter the xylem through the wound or opening at the base of the leaf sheath and interact with the xylem parenchyma cells inside the xylem to enter the vessels.

[0004] The pathogen Xoo of BLB of rice and Xanthomonas oryzae pv. oryzicola (Xoc), the pathogen of bacterial leaf streak of rice, both belong to different pathogenic varieties of Xanthomonas oryzae, and have many similarities in morphological structure, biological characteristics and disease epidemic factors, which brings challenges to the differentiation of the two.

[0005] At present, there is no effective and feasible prevention and control method for rice bacterial leaf blight, and general comprehensive prevention and control measures are usually adopted, which depends on timely and accurate detection of Xoo. Therefore, it is of great significance to establish a rapid and practical detection technology of rice Xoo for preventing and controlling rice bacterial leaf blight. At present, the detection technology for bacterial leaf blight includes traditional observation of disease symptoms, pathogenic bacteria isolation and culture, phage detection and pathogenicity determination, etc. However, the traditional detection method is time-consuming and laborious, and needs to isolate and culture the pathogenic bacteria, and often fails due to the small amount of bacteria carried by the sample or the complex process of isolation and culture, thereby causing missed detection of the pathogenic bacteria. With the development of molecular biology, the molecular detection method based on polymerase chain reaction (PCR) is widely used in the detection of pathogenic bacteria of rice. However, Xoo and Xoc belong to two pathogenic varieties under the same species, and it is difficult to completely distinguish them due to the close genetic relationship and the cross-reaction of PCR detection. Therefore, many studies use double or even multiple PCR to simultaneously detect the two pathogenic bacteria, and specific primers must be designed to identify and distinguish the two pathogenic bacteria. The serological detection method is simple, economical and sensitive, and is suitable for the detection of various pathogenic bacteria. The applicant has established two serological methods of Dot-ELISA and colloidal gold immunostrip for detecting Xoc by using the prepared specific monoclonal antibody for the first time, and successfully applied them to the detection of Xoc in rice samples. However, due to the problems of cross-reaction with other bacteria and low sensitivity, there are few reports on the serological detection method of Xoo. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a hybridoma cell strain secreting anti-rice bacterial leaf blight monoclonal antibody and application of the monoclonal antibody. In order to establish an intuitive, simple and rapid serological detection technology of Xoo, the present application prepares a hybridoma cell strain capable of secreting specific monoclonal antibody against Xoo, and further develops Dot-ELISA and colloidal gold immunostrip serological detection technologies using the monoclonal antibody secreted by the hybridoma cell strain as the detection antibody, which are used for the ultra-sensitive, high-specific and broad-spectrum detection of Xoo in rice. The preparation of the hybridoma cell and the monoclonal antibody secreted by the hybridoma cell and the establishment of the serological method have important application value for the diagnosis and prevention and control of rice bacterial leaf blight and the inspection and quarantine of Xoo.

[0007] The technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a hybridoma cell strain 19C9 secreting anti-rice bacterial leaf blight monoclonal antibody, which can secrete specific monoclonal antibody against rice bacterial leaf blight. The hybridoma cell strain 19C9 was deposited with the China General Microbiological Culture Collection Center on June 24, 2024, and the deposit number is CGMCC No. 46004.

[0009] The second aspect of the present application provides an anti-Xoo monoclonal antibody secreted by the hybridoma cell strain 19C9 of the first aspect. -7 The antibody type and subclass is IgG1, κ light chain, the monoclonal antibody has specific immunoreaction with Xoo, the sensitivity of the Dot-ELISA and the colloidal gold immunochromatographic strip established by using the monoclonal antibody reaches 9.85×10 3 CFU / mL, the sensitivity of detecting the homogenate of the leaf tissue of the rice plant infected with Xoo reaches 1:51200-fold dilution and 1:25600-fold dilution respectively, wherein the dilution fold is calculated by weight / volume, and the unit is g / mL.

[0010] The anti-Xoo monoclonal antibody has specific immunoreaction with Xoo, and does not react with 10 other bacteria including X. oryzae and X. oryzae, and does not have immunoreaction with healthy rice plant tissue.

[0011] The third aspect of the present application provides an application of the anti-Xoo monoclonal antibody of the second aspect in Xoo detection, which is an immunological detection method and an immunological detection kit established by using the monoclonal antibody as a detection antibody.

[0012] Compared with the prior art, the present application has the beneficial effects that: 1) the hybridoma cell strain provided by the present application secretes a large amount of anti-Xoo specific monoclonal antibody, and the Dot-ELISA and CGICS serum detection methods established by using the monoclonal antibody as a detection antibody can quickly, specifically, sensitively and accurately detect Xoo; 2) the monoclonal antibody prepared by using the present application can detect Xoo, and the technical requirements of the operator are low, and expensive equipment such as an electron microscope and a PCR instrument is not needed; 3) the monoclonal antibody prepared by using the present application can be effectively used for field Xoo detection and diagnosis, Xoo port inspection and quarantine, and can be used for epidemiological investigation, resistance breeding, scientific prevention and control and the like. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1Dot-ELISA method for detecting Xoo broad spectrum, specificity and sensitivity analysis results; Figure: (a) Dot-ELISA for detecting Xoo broad spectrum and specificity analysis. A1-10: 10 different strains of Xoo, namely Xoo Y2, Xoo PXO86, Xoo PXO99A, Xoo ScYc, Xoo YN11, Xoo FuJ, Xoo OS198, Xoo HEN11, Xoo G2, Xoo C2; B1-10: 10 control bacteria, respectively, X. oryzae pv. oryzicola RS105, X. oryzae pv. oryzicola JS, Acidovorax oryzae RS1, Acidovorax oryzae RS2, X. albilineans FJ1, X. sacchari ACCC10416, Pantoea ananatis F163, Burkhloderia plantarii ZJ171, X. campestris CGMCC1.3408, Burkholderia glumae Os48; each bacterial suspension (10 7 CFU / mL, 2 μL) was spotted on the NC membrane, and then Dot-ELISA was performed, and the color development was positive; (b) Dot-ELISA for detecting the sensitivity of Xoo. The suspensions of Xoo Y2 strain and Xoc RS105 strain (negative control) were serially diluted by 2 times, and the bacterial suspension (2 μL) of each dilution was spotted on the NC membrane for Dot-ELISA test, and the color development was positive.

[0014] Figure 2 Dot-ELISA and PCR method for detecting Xoo in field rice samples and the sensitivity of detecting diseased leaves; Figure: (a) Dot-ELISA for detecting Xoo in field rice samples. 1-22 are field rice samples collected in Zhuji City, Zhejiang Province, "-" is a non-infected rice leaf as a negative control, "+" is an Xoo-infected rice leaf as a positive control; the homogenate of rice leaf tissue was diluted by 20 times, and the homogenate (2 μL) was spotted on the NC membrane, and then Dot-ELISA was performed, and the color development was Xoo positive; (b) PCR for detecting Xoo in field rice samples. The samples are the same as in (a); (c) Dot-ELISA for detecting the sensitivity of Xoo-infected rice leaf tissue homogenate. The homogenate of Xoo-infected rice leaf tissue or non-infected rice leaf was diluted by 2 times, and 2 μL of the homogenate of each dilution was spotted on the NC membrane for detection, and the color development was positive.

[0015] Figure 3The results of the analysis of the broad spectrum, specificity and sensitivity of the colloidal gold immunochromatographic test strip for detecting Xoo are shown in the figure: (a) The results of the analysis of the broad spectrum and specificity of the colloidal gold immunochromatographic test strip for detecting Xoo. A1-10: 10 different strains of Xoo, namely Xoo Y2, Xoo PXO86, Xoo PXO99A, Xoo ScYc, Xoo YN11, Xoo FuJ, Xoo OS198, Xoo HEN11, Xoo G2, Xoo C2; B1-10: 10 control bacteria, which are X. oryzae pv. oryzicola RS105, X. oryzae pv. oryzicola JS, Acidovorax oryzae RS1, Acidovorax oryzae RS2, X. albilineans FJ1, X. sacchari ACCC 10416, Pantoea ananatis F163, Burkhloderia plantarii ZJ171, X. campestris CGMCC 1.3408, Burkholderia glumae Os48; each bacterial suspension (10 7 CFU / mL, 100 μL) was detected, and both T line and C line showed positive coloration, and only C line showed negative coloration; (b) Sensitivity of the colloidal gold immunochromatographic test strip for detecting Xoo suspension. The suspensions of Xoo Y2 strain and Xoc RS105 strain (negative control) were serially diluted by multiple times, and each diluted suspension (100 μL) was dropped onto the sample pad for detection. (c) Sensitivity of the colloidal gold immunochromatographic test strip for detecting Xoo-infected rice leaf tissue homogenate. The homogenate of Xoo-infected rice leaf tissue was diluted by multiple times, and 100 μL of the homogenate at each dilution was dropped onto the sample pad for detection, and the uninfected rice leaf was used as a control. The samples showing coloration of both T line and C line were positive for Xoo infection.

[0016] BIOLOGICAL DEPOSIT

[0017] The monoclonal antibody hybridoma cell strain 19C9 secreting an antibody against Xoo was deposited with the China General Microbiological Culture Collection Center on June 24, 2024, at the address of No. 1, Beichen West Road, Chaoyang District, Beijing, with the postal code of 100101, and the deposit number of CGMCC No. 46004. DETAILED DESCRIPTION

[0018] The monoclonal antibody hybridoma cell strain 19C9 secreting an antibody against Xoo was deposited with the China General Microbiological Culture Collection Center on June 24, 2024, at the address of No. 1, Beichen West Road, Chaoyang District, Beijing, with the postal code of 100101, and the deposit number of CGMCC No. 46004.

[0019] An anti-Xoo monoclonal antibody secreted by the hybridoma cell strain 19C9, the indirect ELISA titer of the monoclonal antibody in ascites is 10 -7 The antibody type and subclass is IgG1, kappa light chain, the monoclonal antibody has specific immunoreaction with Xoo, the sensitivity of the Dot-ELISA and colloidal gold immunostrip established by using the monoclonal antibody for detecting Xoo reaches 9.85x10 3 CFU / mL, the sensitivity of detecting the homogenate liquid of the leaf tissue of the rice plant infected with Xoo reaches 1:51200-fold dilution and 1:25600-fold dilution (the dilution fold is weight / volume, unit: g / mL) respectively.

[0020] The anti-Xoo monoclonal antibody has specific immunoreaction with Xoo, and does not react with 10 other bacteria including X. oryzae and X. oryzae pv. oryzae, and does not have immunoreaction with healthy rice plant tissue.

[0021] The anti-Xoo monoclonal antibody is applied to the detection of the bacteria, and various immunological detection methods and immunological detection kits established by using the monoclonal antibody as a detection antibody.

[0022] The hybridoma cell strain provided by the application can secrete a large amount of anti-Xoo monoclonal antibody, and the secreted monoclonal antibody has high specificity, high sensitivity, high titer and good stability. The high-throughput serological method for detecting Xoo established by using the monoclonal antibody as a detection antibody can be applied to the detection and diagnosis of Xoo in the field rice plant sample and the inspection and quarantine at the port, so as to provide reagents and technical support for the scientific prevention and control of Xoo in China.

[0023] The application will be further described below in combination with examples and drawings.

[0024] I. Establishment of hybridoma cell and preparation of monoclonal antibody thereof

[0025] 1. Materials

[0026] The strains used in this example and their sources are shown in Table 1. The infected Xoo or healthy rice plants were collected in 2023 in the rice field of Zhuji City, Zhejiang Province. Polyethylene glycol, hypoxanthine, aminopterin and thymidine additive (hypoxanthine-aminopterin-thymidine media supplement, HAT), goat anti-mouse secondary antibody labeled with AP, monoclonal antibody type and subclass identification kit were purchased from Sigma-Aidrich Company (USA); NBT / BCIP color developing substrate was a product of Promega Company (USA); nitrocellulose membrane (NC membrane) and Protein A-Agarose were purchased from GE healthcare Company (USA); 96-well enzyme-labeled plate and cell plate were purchased from Corning Company (USA). SPF BALB / c mice were purchased from Shanghai Xipu-Bikai Experimental Animal Co., Ltd.

[0027] Table 1 Strains used in this example and their sources

[0028]

[0029] 2. Strain culture and PCR identification

[0030] The strains were streaked on nutrient agar (NA) medium and incubated at 28°C for 24 hours. After colony PCR verification, single colonies were transferred to NA liquid medium and incubated at 28°C, 200 rpm for 10 hours. The obtained bacterial culture was centrifuged at 13500 x g for 5 minutes, resuspended in 1 ml sterile water, and the bacterial concentration was determined using the standard plate counting procedure.

[0031] 3. Preparation of immunogen

[0032] The Xoo Y2 strain was cultured in liquid NA medium at 28°C for 24 hours, then centrifuged at 13500 x g for 5 minutes. The obtained precipitate was resuspended in a physiological saline solution containing 0.5% formaldehyde, and the bacteria were inactivated by incubation at 4°C for 24 hours. After washing twice with physiological saline, the inactivated bacteria were used as immunogen.

[0033] 4. Immunization of animals

[0034] The inactivated Xoo Y2 strain (10 7 CFU per mouse) was mixed and emulsified with an equal amount of Freund's complete adjuvant and injected into the abdominal cavity of 8-week-old female BALB / c mice. After 3 weeks, the same inactivated bacteria were emulsified with Freund's incomplete adjuvant for the second booster immunization, and after another 3 weeks, the inactivated bacteria (2 x 10 7 CFU per mouse) were diluted with physiological saline and injected into the abdominal cavity of the mice.

[0035] 5. Cell fusion

[0036] The spleen cells of the immunized mice and mouse myeloma cells Sp2 / 0 were mixed at a ratio of 5:1 in serum-free RPMI 1640 (Gibco) medium, and after centrifugation at 1500 rpm for 5 minutes, the supernatant medium was removed. Then, 1 mL of 50% PEG (molecular weight 1500) fusion agent was added to the centrifuge tube containing the cells, and the tube was placed in a 37°C water bath for 2 minutes. The fusion was terminated with serum-free RPMI 1640 medium, and after centrifugation at 1500 rpm for 5 minutes, the supernatant was removed. The precipitate was suspended in RPMI 1640 medium containing HAT, fetal bovine serum, and ampicillin, and was dispensed into 96-well cell plates. The plates were incubated in a 37°C, 5% CO2 cell incubator and screened for hybridoma cells.

[0037] 6. Screening of hybridoma cells and positive wells, and cell cloning

[0038] The fusion rate of the cells reached 100%, and 10 days after fusion, when the hybrid cells were cultured to cover more than one-tenth of the bottom of the well, indirect ELISA was used to detect Xoo antibodies in the cell culture supernatant. The results showed that the Xoo antibodies in the supernatant of 153 cell wells were positive. The 153 positive wells were filled with medium, and after one day, the specificity and sensitivity of the antibodies secreted by the positive wells were analyzed. Three hybridoma cells with high sensitivity and specificity were subjected to cell cloning, i.e., the cells were blown up with HT medium, and the cells were aspirated and gradient-diluted in the leftmost column of wells in a 96-well plate. Under a microscope, wells containing about 100 cells were selected, and an appropriate amount of HT medium was used to blow and distribute the cells evenly into new 96-well cell plates for culture. After 3-5 days, the plates were observed under a microscope, and wells containing only monoclonal cells were selected and filled with HT medium. After 5 days, the specificity and sensitivity of the antibodies in the supernatant of the wells were detected. Monoclonal cells with high sensitivity and specificity were selected and subjected to the above-mentioned cloning, culture, and screening. After three consecutive clonings, a hybridoma cell line 19C9 secreting specific monoclonal antibodies against Xoo was finally obtained.

[0039] The hybridoma cell line 19C9 was deposited with the China General Microbiological Culture Collection Center on June 24, 2024, at address No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, China, with a postal code of 100101, and the deposit number was CGMCC No. 46004.

[0040] 7. Preparation and purification of monoclonal antibody ascites

[0041] Twelve-week-old BALB / c male mice were injected intraperitoneally with 200 μl of pristane, and after 7-10 days, about 7×10 5The hybridoma cells of 19C9 were injected into the abdominal cavity of BALB / c mice. The mice were killed 6-10 days after injection. The ascites was collected by needle and centrifuged at 8000 rpm for 3 minutes. The supernatant was collected and was the monoclonal antibody ascites. A total of 30 mL of ascites was obtained.

[0042] The ascites was diluted with 2 volumes of physiological saline. Saturated ammonium sulfate solution (pH 7.2) was added dropwise while stirring at room temperature. After stirring for 10 minutes, it was left to stand at 4°C overnight. It was centrifuged at 12000 rpm for 10 minutes. The supernatant was discarded. The precipitate was suspended in 2 mL of PBS. It was dialyzed in PBS pre-cooled at 4°C for 24 hours. The PBS was changed every 4 hours during the dialysis. The purified monoclonal antibody IgG was obtained after dialysis. It was stored in a refrigerator at -80°C.

[0043] 8. Subclass identification and ascites titer determination of monoclonal antibody

[0044] The purified monoclonal antibody ascites was subjected to DAS-ELISA identification according to the kit instructions using standard anti-BALB / c mouse IgG1, IgG2a, IgG2b, IgG3, IgM, λ, κ antibodies from Sigma. 2a 2b The results showed that the subclass of 19C9 monoclonal antibody was IgG1, κ light chain. Xoo (1×10 7 CFU / mL) was used as the antigen. Xoo was used as the coating antigen. The 19C9 monoclonal antibody ascites titer was determined by indirect ELISA. The analysis results showed that the titer of the monoclonal antibody ascites was 10 -7 .

[0045] 9. ACP-ELISA method for analyzing the specificity of monoclonal antibody

[0046] ​Ten different isolates of Xoo (Xoo Y2, Xoo PXO86, Xoo PXO99A, Xoo ScYc, Xoo YN11, Xoo FuJ, Xoo OS198, Xoo HEN11, Xoo G2, Xoo C2), five bacteria of the same genus of Xoo (X. oryzae pv. oryzicola RS105, X. oryzae pv. oryzicola JS, X. albilineans FJ1, X. sacchari ACCC 10416, X. campestris CGMCC 1.3408) and five bacteria infecting rice (Acidovorax oryzae RS1, Acidovorax oryzae RS2, Pantoea ananatis F163, Burkhloderia plantarii ZJ171 and Burkholderia glumae Os48) were used as test samples, and Xoo-infected rice leaf tissues and healthy rice tissues were used as positive and negative controls, respectively, to analyze the specificity of 19C9 mAb by ACP-ELISA method. The steps of ACP-ELISA method were as follows: 100 μL / well of the bacterial suspension (10 7 CFU / mL) or rice plant tissue homogenate (1:20 dilution) was coated on an ELISA plate at 4°C overnight or 37°C for 4 h; after washing with PBST for 3 times, 3% skim milk was added for blocking for 30-40 min; 100 μL / well of 1:5000 diluted mAb ascites was added and incubated at 37°C for 1 h; after washing with PBST for 3 times, 100 μL / well of 1:8000 diluted alkaline phosphatase (AP)-labeled rabbit anti-mouse IgG secondary antibody (Sigma) was added and incubated at 37°C for 1 h; after washing with PBST for 4 times, PNPP substrate was added for color development for 20-30 min; after the reaction was terminated by 2 mol / L sodium hydroxide, the OD 405 value was read by a microplate reader, and the samples with the ratio of OD value to negative OD value greater than 3.0 were positive. It was found that 19C9 mAb had strong positive immune reaction with 10 Xoo strains and Xoo-infected rice tissues, but had no reaction with other 10 plant bacteria.

[0047] II. Establishment of serological method for detecting Xoo

[0048] 1. Establishment of Dot-ELISA detection method

[0049] 1.1 Detection steps of Dot-ELISA method

[0050] 1) 100 μL / well of 1 × 10 7CFU / mL), and the liquid nitrogen-grounded homogenate of the Xoo-infected rice tissue as the detection sample;

[0051] 2) Spotting: 2 μL of the sample was spotted onto a nitrocellulose (NC) membrane, and healthy and Xoo-infected rice leaves were set as negative and positive controls, respectively, and dried in a 37°C incubator for 10 minutes;

[0052] 3) The NC membrane was blocked in a 5% skim milk powder-containing PBST (0.01M PBS containing 0.05% Tween-20) blocking solution at room temperature for 30 minutes. Then, the NC membrane was incubated in a moderately diluted 19C9 monoclonal antibody at room temperature for 1 hour;

[0053] 5) Membrane washing: the membrane was washed with PBST for 3 times, each for 3 minutes;

[0054] 6) The NC membrane was incubated in a moderately diluted AP-labeled goat anti-mouse IgG secondary antibody at room temperature for 1 hour, and then washed with PBST for 3 times, each for 3 minutes, and washed with PBS once;

[0055] 7) Color developing solution was prepared by mixing 66 μL of NBT and 33 μL of BCIP substrate (Promega) into 10 mL of substrate buffer (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl, pH 9.5).

[0056] 8) After the NC membrane was dried with a water-absorbing paper, it was placed in the substrate solution for reaction, and color development was observed at room temperature for 15-20 minutes. The results were observed by naked eyes, and when the positive control showed obvious purple spots and the negative control showed no color change, the reaction was terminated by rinsing the membrane in tap water, and the results were photographed and recorded.

[0057] 1.2 Establishment of the Xoo Dot-ELISA method

[0058] The Xoo monoclonal antibody and the AP-labeled goat anti-mouse IgG secondary antibody were diluted by 1:1000, and the optimal working concentrations of the monoclonal antibody and the enzyme-labeled secondary antibody in the Dot-ELISA method were determined by square array experiments. The experimental results showed that the optimal working concentrations of the monoclonal antibody and the enzyme-labeled secondary antibody were 1:5000 and 1:8000, respectively. The Dot-ELISA method for detecting Xoo in plants was established based on the optimal working concentrations of the above antibodies.

[0059] 1.3 Specificity, broad spectrum and sensitivity of the Dot-ELISA method for detecting Xoo

[0060] Ten different isolates of Xoo (Xoo Y2, Xoo PXO86, Xoo PXO99A, Xoo ScYc, Xoo YN11, Xoo FuJ, Xoo OS198, Xoo HEN11, Xoo G2, Xoo C2), five bacteria of the same genus of Xoo (X. oryzae pv. oryzicola RS105, X. oryzae pv. oryzicola JS, X. albilineans FJ1, X. sacchari ACCC 10416, X. campestris CGMCC 1.3408) and five bacteria infecting rice (Acidovorax oryzae RS1, Acidovorax oryzae RS2, Pantoea ananatis F163, Burkhloderia plantarii ZJ171 and Burkholderia glumae Os48) were used as detection samples to analyze the specificity and broad spectrum of Dot-ELISA method for detecting Xoo. The detection results showed that the established Dot-ELISA method could specifically and broadly detect the 10 Xoo strains, and had no cross-reaction with the 10 non-Xoo strains including Xoc (bacteria of bacterial leaf streak of rice) Figure 1 a).

[0061] The bacterial liquid (5 x 10 6 CFU / mL) of Xoo Y2 strain and Xoc RS105 strain (negative control) were serially diluted by 10 times and then spotted to analyze the sensitivity of Dot-ELISA method for detecting Xoo. The detection results showed that the sensitivity of the established Dot-ELISA method for detecting Xoo reached 9.85 x 10 3 CFU / mL Figure 1 b).

[0062] The established Dot-ELISA method was used to detect Xoo in 22 actual leaf samples, and healthy rice leaves were used as negative control and Xoo bacterial liquid as positive control. It was found that 17 samples produced positive reaction Figure 2 a). The samples were simultaneously detected and analyzed by PCR method, and the results showed that Xoo specific gene fragments were amplified in all serological positive samples, and Xoo specific gene fragments were not amplified in all serological negative samples Figure 2 b). PCR product nucleic acid sequencing and sequence alignment analysis showed that the PCR positive samples were indeed infected with Xoo, indicating that the detection results of serological method were completely consistent with PCR, indicating that Dot-ELISA method could be accurately and reliably used for detection of Xoo in rice.

[0063] The sensitivity of the Dot-ELISA method for detecting the homogenate of infected leaves was analyzed by diluting the infected rice tissues with Xoo from 1:25 to 1:51200 times (weight / volume, g / mL), and the same dilution was also performed on healthy rice leaves. The results showed that the sensitivity of the Dot-ELISA method for detecting the homogenate of infected leaves with Xoo reached 1:51200 times (weight / volume, g / mL) (c) in the above. Figure 2 The results showed that the Dot-ELISA method had high sensitivity for detecting Xoo in rice.

[0064] 2. Preparation of colloidal gold immunodetection test strips

[0065] 2.1 Preparation and detection steps of Xoo colloidal gold immunodetection test strips

[0066] Colloidal gold particles were prepared by citrate reduction method. 200 mL dd H2O and 2 mL 1% chloroauric acid were added to a conical flask, stirred and heated to boiling, 4 mL 1% sodium citrate was quickly added, and heating was continued for 15-20 minutes until it turned dark red, then cooled at room temperature, and stored at 4°C. The pH value of the blank colloidal gold solution was adjusted to 7.4 with 0.1M K2CO3, and a suitable concentration of antibody was added dropwise under stirring, 30 μg of 19C9 monoclonal antibody was added per 1 mL of colloidal gold, and 1 mL of 5% BSA was added after stirring for 30 minutes, and then resuspended with gold-labeled antibody storage solution (0.01M PBS) after centrifugation;

[0067] The glass fiber sample pad was soaked in 0.01M PBS containing 1% BSA and 0.05% Tween-20 for 30 minutes, and then dried at 37°C. The colloidal gold-labeled monoclonal antibody 19C9 was sprayed onto the colloidal gold-binding pad, and dried at 37°C for 2 hours. The Bio-Dot XYZ-3000 dot membrane gold spraying instrument (Bio-Dot, CA, USA) was used to spray the capture monoclonal antibody 19C9 and goat anti-mouse antibody onto the test line (T) and control line (C) on the nitrocellulose membrane (NC) membrane, respectively, and dried at 37°C for 2 hours;

[0068] All the above sample pads, colloidal gold-binding pads, nitrocellulose membranes, and water-absorbing pads were assembled on a polyvinyl chloride backboard, with an overlap of 2 mm at each connection. The assembled plate was cut longitudinally into 3 mm wide strips, and the card plate was clamped tightly, then sealed in a bag for use.

[0069] Operation steps of colloidal gold immunodetection test strips for plant samples:

[0070] a. After the rice plant tissue was weighed and ground, 0.01M PBS (pH 7.4) was added at a ratio of 1:20-1:50 (w / v, g / mL) to homogenize the grinding;

[0071] b. Spotting: One of the above colloidal gold immunochromatographic test strips was taken from the aluminum foil bag and laid flat, and the rice plant sample homogenate was taken with a plastic pipette and 3-4 drops (about 100-200 μL) were dropped into the sample well of the colloidal gold immunochromatographic test strip;

[0072] c. Visual observation to determine the results: the results were visually determined within 5-10 minutes, i.e. the sample with red bands appearing on both T and C lines was positive, the sample with red bands appearing on the C line but not on the T line was negative. If neither the C nor the T line appeared a red band, or only the T line appeared a red band but not the C line, it indicated that the test strip was invalid.

[0073] 2.2 Specificity, broad spectrum and sensitivity of the colloidal gold immunochromatographic test strip for detecting Xoo

[0074] Ten different isolates of Xoo (Xoo Y2, Xoo PXO86, Xoo PXO99A, Xoo ScYc, Xoo YN11, Xoo FuJ, Xoo OS198, Xoo HEN11, Xoo G2, Xoo C2), five bacteria of the same genus as Xoo (X. oryzae pv. oryzicola RS105, X. oryzae pv. oryzicola JS, X. albilineans FJ1, X. sacchari ACCC 10416, X. campestris CGMCC 1.3408) and five bacteria infecting rice (Acidovorax oryzae RS1, Acidovorax oryzae RS2, Pantoea ananatis F163, Burkhloderia plantarii ZJ171 and Burkholderia glumae Os48) were used as detection samples to analyze the specificity and broad spectrum of the colloidal gold immunochromatographic test strip for detecting Xoo. The detection results showed that the prepared colloidal gold immunochromatographic test strip could specifically and broadly detect the 10 Xoo strains, and had no cross-reaction with the 10 non-rice bacterial blight strains including Xoc (Xanthomonas oryzae pv. oryzae) (a) in the present application. Figure 3

[0075] Xoo Y2 bacterial solution (2.5 x 10 6 CFU / mL) was diluted by a certain ratio and spotted to analyze the sensitivity for detecting Xoo. The detection results showed that the sensitivity of the prepared colloidal gold immunochromatographic test strip for detecting Xoo reached 9.85 x 10​3 CFU / mL Figure 3 b) in the

[0076] The sensitivity of colloidal gold immunochromatographic strip for detecting infected leaves was analyzed by diluting the homogenate of infected leaves with Xoo from 1:25 to 1:25600 (w / v, g / mL) using 0.01M PBS, and using 1:25 dilution of the homogenate of healthy rice leaf tissues as negative control. The analysis results showed that the sensitivity of the prepared colloidal gold immunochromatographic strip for detecting the homogenate of infected leaves with Xoo reached 1:25600 dilution (w / v, g / mL) Figure 3 c) in the

[0077] 3. Rice bacterial leaf blight Dot-ELISA detection kit

[0078] 1) Main components of the kit:

[0079]

[0080] The above reagents are stored at 4°C

[0081] Nitrocellulose membrane (NC) 10 sheets

[0082] Skimmed milk powder 30g

[0083] 10x PBST 1 bottle 100mL

[0084] Substrate buffer 1 bottle 100mL

[0085] 2) Operation steps for detecting plant samples:

[0086] a. After weighing the rice plant tissues, grind them, add 0.01M PBS (pH 7.4) at a ratio of 1:20-1:50 (w / v, g / mL), and homogenize;

[0087] b. Take 2μL of the supernatant and apply it to the NC membrane, and set healthy and infected Xoo plant tissues as negative and positive controls, respectively, dry at room temperature for 5-10 minutes;

[0088] c. Immerse the NC membrane in PBST containing 5% skimmed milk powder for blocking at room temperature for 30 minutes;

[0089] d. Place the NC membrane in 1:5000 dilution of the monoclonal antibody and incubate at room temperature for 1 hour;

[0090] e. Wash the membrane with PBST for 3-4 times, each for 3 minutes; place the NC membrane in 1:8000 dilution of AP enzyme-labeled goat anti-mouse IgG secondary antibody and incubate at room temperature for 1 hour;

[0091] f. Wash the membrane with PBST for 4 times, each for 3 minutes;

[0092] g.66 μL NBT and 33 μL BCIP substrate were added into 10 mL substrate buffer and mixed well, the membrane was put into the solution for color development for 15-20 minutes. When the positive control showed obvious purple color and the negative control showed no color, the reaction was terminated by running tap water to rinse the membrane and the result was recorded by taking a photo.

[0093] 3) Storage and shelf life:

[0094] Store at 2-8°C in the dark, shelf life 12 months.

[0095] 4) 0.01M Phosphate Buffer Solution (PBS pH 7.4) formula:

[0096]

[0097] Add distilled water 950 mL, adjust pH to 7.4, and dilute to 1000 mL.

[0098] 4. Colloidal gold immunochromatographic strip test kit for Xanthomonas oryzae pv. oryzae

[0099] 1) Main components of the kit:

[0100]

[0101] The above reagents are stored at 4°C

[0102] 2) Operation steps for detecting plant samples:

[0103] a. After weighing the rice plant tissue, grind it and add 0.01M PBS (pH 7.4) at a ratio of 1:20-1:50 (w / v, g / mL) to homogenize the sample;

[0104] b. Spotting: Take one colloidal gold immunochromatographic strip from the aluminum foil bag and lay it flat. Use a plastic pipette to draw 3-4 drops (about 100-200 μL) of the rice plant homogenate and drop it into the sample well of the colloidal gold immunochromatographic strip;

[0105] c. Visual observation to determine the result: The result can be determined visually within 5-10 minutes. If both the T line and the C line show red bands, the sample is positive. If only the C line shows a red band and the T line does not, the sample is negative. If neither the C line nor the T line shows a red band, or only the T line shows a red band and the C line does not, the test strip is invalid.

[0106] The above-described embodiments are only the preferred ones of the present application, and are not intended to limit the present application. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application. Therefore, any technical solutions obtained by equivalent replacement or equivalent transformation shall fall within the protection scope of the present application.

Claims

1. A hybridoma cell line 19C9 secreting anti-Xanthomonas oryzae pv. oryzae monoclonal antibody, characterized by The hybridoma cell strain 19C9 capable of secreting the anti-Xanthomonas oryzae pv. oryzae monoclonal antibody was preserved in China General Microbiological Culture Collection Center on June 24, 2024, and the preservation number is CGMCC No. 46004.

2. The anti-Xanthomonas oryzae pv. oryzae monoclonal antibody secreted by the hybridoma cell strain 19C9 according to claim 1.

3. The anti-Xanthomonas campestris pv. oryzae monoclonal antibody of claim 2, wherein The indirect ELISA titer of the ascites of the monoclonal antibody reached 10 -7 The antibody type and subclass is IgG1, κ light chain, the monoclonal antibody has specific immunoreaction with Xoo, the sensitivity of the Dot-ELISA and colloidal gold immunostrip established by using the monoclonal antibody to detect Xoo reaches 9.85×10 3 CFU / mL, the sensitivity of the detection of the homogenate of the leaf tissue of the rice plant infected with Xoo reaches 1:51200-fold dilution and 1:25600-fold dilution respectively, wherein the dilution fold is calculated by weight / volume, and the unit is g / mL.

4. The anti-Xoo mAb of claim 2, wherein the Xoo is Xoo race T1. The monoclonal antibody can specifically react with Xanthomonas oryzae pv. oryzae, and does not react with 10 other bacteria including Xanthomonas campestris and Xanthomonas oryzae pv. oryzae, and does not react with healthy rice plant tissues.

5. The use of monoclonal antibody against Xanthomonas campestris pv. oryzae of claim 2 for detecting Xanthomonas campestris pv. oryzae, characterized in that The application is various immunological detection methods and immunological detection kits established by taking the anti-Xanthomonas oryzae pv. oryzae monoclonal antibody as a detection antibody.

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