Monoclonal antibody for detecting sugarcane yellow leaf virus and application and detection method thereof

Abstract

The invention relates to the technical field of biology, in particular to a monoclonal antibody for detecting sugarcane yellow leaf virus as well as application and a detection method thereof. A nucleotide sequence of a variable region sequence of a light chain of the monoclonal antibody is as shown in SEQ ID No.1, and an amino acid sequence is as shown in SEQ ID No.2; a variable region sequence of a heavy chain of the monoclonal antibody is as shown in SEQ ID No.3, and an amino acid sequence is as shown in SEQ ID No.4. The monoclonal antibody for detecting the sugarcane yellow leaf virus is obtained, and variable region sequences of light and heavy chains of the antibody are directly detected. Compared with the monoclonal antibody prepared by the traditional hybridoma, the degradation, mutation or loss of the monoclonal antibody is not worried about. According to the invention, a monoclonal antibody of the sugarcane yellow leaf virus is taken as a primary antibody, and a detection method is established to detect the sugarcane yellow leaf virus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a monoclonal antibody for detecting sugarcane yellow leaf virus and an application and a detection method thereof. Background Art

[0002] Sugarcane (Saccharum spp. hybrids) is the largest sugar crop cultivated in my country, and its yield directly impacts the country's sugar security. In China's sugarcane-producing regions, long-term continuous cropping, perennial perennials, and a monoculture system, coupled with recent climate diversification, have significantly increased the likelihood of plant disease during growth. Sugarcane yellow leaf disease, caused by the sugarcane yellow leaf virus (SCYLV), is a contagious disease that causes premature aging of sugarcane, leading to reduced yield and sucrose content. Severe cases of SCYLV infection have been reported to reduce sugarcane yield by up to 60%. This disease is threatening my country's sugarcane industry.

[0003] In production, sugarcane yellow leaf disease (YLV) can be easily confused with sugarcane chlorosis in the early and even late stages of disease. Identifying plants infected with YLV solely based on phenotypic traits requires extensive experience and is prone to missed or misidentified infections. Therefore, molecular or immunoassay techniques are needed for differentiation and confirmation. Currently, the primary detection techniques used for sugarcane yellow leaf disease (YLV) are RT-PCR (Reverse transcription-polymerase chain reaction) and real-time fluorescence quantitative PCR (qRT-PCR). However, both methods require expensive equipment and specialized technicians, resulting in high costs, cumbersome procedures, and low efficiency. Therefore, phenotypic and molecular-based YLV detection in production is not suitable for the large-scale sugarcane breeding population (millions of seedlings planted annually). For sugarcane breeding and production, there is an urgent need for a rapid, convenient, stable, low-cost identification technology suitable for large-scale testing during the early stages of sugarcane growth, such as the seedling or jointing stages. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a monoclonal antibody for detecting sugarcane yellow leaf virus, its application and detection method. The present invention uses Single B rapid monoclonal antibody based on the sequence of CP gene to prepare a monoclonal antibody that can specifically identify sugarcane yellow leaf disease, and establishes a rapid and low-cost sugarcane yellow leaf disease detection technology based on this antibody. The monoclonal antibody provided by the present invention can specifically detect sugarcane yellow leaf virus.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a monoclonal antibody for detecting sugarcane yellow leaf virus. The nucleotide sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID No. 1, and the amino acid sequence is shown as SEQ ID No. 2; the variable region sequence of the heavy chain of the monoclonal antibody is shown as SEQ ID No. 3, and the amino acid sequence is shown as SEQ ID No. 4.

[0007] Preferably, the monoclonal antibody is prepared by immunizing an animal with the CP protein encoded by the CP gene.

[0008] Preferably, the nucleotide sequence of the CP gene is shown as SEQ ID No.5.

[0009] The present invention also provides the use of the monoclonal antibody described in the above technical solution in detecting sugarcane yellow leaf virus.

[0010] The present invention also provides a method for detecting sugarcane yellow leaf virus, comprising the following steps:

[0011] 1) Take the basal vein of a sugarcane leaf, cut it into a flat surface, and press the cut surface onto a nitrocellulose membrane to obtain a nitrocellulose membrane with an imprinted spot;

[0012] 2) placing the nitrocellulose membrane with the blot obtained in step 1) into a TBST buffer solution containing 2% by weight skim milk for blocking to obtain a blocked nitrocellulose membrane;

[0013] 3) immersing the blocked nitrocellulose membrane obtained in step 2) in a TBST buffer solution containing 1% by weight skim milk, and then mixing with the monoclonal antibody according to any one of claims 1 to 3, and incubating with the primary antibody;

[0014] 4) discarding the liquid and washing with TBST buffer to obtain a washed nitrocellulose membrane;

[0015] 5) Immersing the washed nitrocellulose membrane obtained in step 4) in TBST buffer containing 1% by weight skim milk powder, and then mixing with AP-labeled rabbit anti-human IgG antibody for secondary antibody incubation;

[0016] 6) discarding the liquid and washing with TBST buffer to obtain a washed nitrocellulose membrane;

[0017] 7) mixing the washed nitrocellulose membrane obtained in step 6) with a BCIP / NBT color developing solution, developing color for 30-35 minutes, and terminating the reaction. When the blot on the nitrocellulose membrane turns purple, the sugarcane leaves are infected with sugarcane yellow leaf virus; when the blot on the nitrocellulose membrane does not turn purple, the sugarcane leaves are not infected with sugarcane yellow leaf virus.

[0018] The volume ratio of the number of blots contained in the washed nitrocellulose membrane to the volume of BCIP / NBT color development solution was 1:100 μL.

[0019] Preferably, the pressing time in step 1) is 10 to 15 seconds;

[0020] The blot on the nitrocellulose membrane with the blot is air-dried and then blocked;

[0021] The specifications of the nitrocellulose membrane are 6 cm in length and 5 cm in width, and the pore size is 0.45 μm.

[0022] Preferably, in step 2), the volume ratio of the nitrocellulose membrane with the blotted spot to the TBST buffer is 1:35 mL;

[0023] The blocking conditions include: blocking at 20-30° C. for 1 hour.

[0024] Preferably, the monoclonal antibodies in step 3) are mixed in the form of a monoclonal antibody solution with a concentration of 2.7 mg / mL;

[0025] The volume ratio of the blocked nitrocellulose membrane, the volume of TBST buffer, and the monoclonal antibody solution was 1:35 mL:4.6 μL;

[0026] The primary antibody incubation conditions include: incubation at a temperature of 20-30° C. for 3 hours and a rotation speed of 15 rpm.

[0027] Preferably, the washing conditions in steps 4) and 6) include: a rotation speed of 40 rpm, two washes, each time for 5 minutes.

[0028] Preferably, in step 5), the AP-labeled rabbit anti-human IgG antibody is mixed in the form of an AP-labeled rabbit anti-human IgG antibody solution at a concentration of 0.2 mg / ml;

[0029] The volume ratio of the washed nitrocellulose membrane to the volume of TBST buffer and AP-labeled rabbit anti-human IgG antibody solution is 1:35 mL:10 μL;

[0030] The secondary antibody incubation conditions include: incubation at 4° C. overnight and a rotation speed of 15 rpm.

[0031] The entire genome of sugarcane yellow leaf virus (SYLV) is approximately 6.0 kb in size. 72% of the virus is protein, with the remainder being nucleic acid. It has six well-defined open reading frames (ORFs) and three untranslated regions (UTRs). ORF3 encodes the viral coat protein (CP), which is involved in the assembly of the viral coat and viral RNA transcription. Because the region encoding the CP in ORF3 is relatively conserved, it is often used as a primer design in molecular detection. In my country, researchers have generated polyclonal and monoclonal antibodies using the CP gene sequence. However, polyclonal antibodies lack the specificity of monoclonal antibodies, resulting in inferior detection performance. Currently, monoclonal antibodies that recognize SYLV are produced using traditional hybridoma antibody production techniques, which can lead to hybridoma degeneration, mutation, or loss during use. Therefore, the present invention utilizes a rapid Single B monoclonal antibody production method to generate monoclonal antibodies that specifically recognize SYLV based on the CP gene polypeptide sequence and directly detect the variable region sequences of the antibody's light and heavy chains. There is no need to worry about the degeneration, mutation or loss of hybridomas during use.

[0032] Since the main part of the infection site of sugarcane yellow leaf virus is the vein part of the leaf, and the vein of sugarcane is hard, it can be directly imprinted onto a nitrocellulose membrane to adsorb the virus particles. The virus particles are then bound to the membrane using a specific monoclonal antibody that recognizes the coat protein and is prepared based on Single B rapid monoclonal antibody. The membrane is then incubated with an antibody that is compatible with the specific monoclonal antibody and labeled with alkaline phosphatase (AP). Finally, the membrane is stained using a BCIP / NBT display kit to observe the results.

[0033] Finally, the present invention uses Single B rapid monoclonal antibody based on the sequence of CP gene to prepare a monoclonal antibody that can specifically identify sugarcane yellow leaf disease, and based on this antibody, establishes a low-cost method for rapid detection and determination of sugarcane yellow leaf virus disease and applies it.

[0034] Beneficial effects of the present invention:

[0035] 1. The present invention obtains monoclonal antibodies for detecting sugarcane yellow leaf virus and directly detects the variable region sequences of the antibody's light and heavy chains. Compared with monoclonal antibodies prepared from traditional hybridomas, there is no need to worry about monoclonal antibody degradation, mutation, or loss.

[0036] 2. The present invention uses a monoclonal antibody against sugarcane yellow leaf virus as a primary antibody to establish a detection method for detecting sugarcane yellow leaf virus.

[0037] 3. The detection method established by the present invention is easy to operate and does not require expensive equipment. It can detect 300 samples per day with a cost as low as RMB 2 per sample. Its high efficiency and low cost are highly favored by people.

[0038] 4. The detection method established by the present invention can also accurately detect virus-infected leaves without phenotypic symptoms.

[0039] 5. The detection method established in this invention is not only suitable for the detection of small samples in the laboratory, but also for the rapid detection of large-scale samples in the field. It provides a supporting technology for the production of virus-free sugarcane seedlings and the identification of disease resistance in large sugarcane breeding populations, and thus has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0041] Figure 1 Specificity and sensitivity of sugarcane yellow leaf virus (SCYLV) detected using a monoclonal antibody-based tissue blot immunoassay (TBIA); A: Phenotypic comparison of sugarcane leaves infected with SCYLV (left) and healthy leaves (right); B: TBIA results: purple spots indicate the presence of SCYLV in infected leaves (left), while no signal was observed in healthy leaves (right); C: Sensitivity analysis of purified SCYLV coat protein; serial dilutions (0.6 mg / mL to 3 pg) showed a detection limit of 30 pg; DH: Specificity evaluation: TBIA results of leaves infected with sugarcane mosaic virus (SCMV; D), sorghum mosaic virus (SrMV; E), sugarcane streak mosaic virus (SCSMV; F), a negative control (NTC; G), and a SCYLV-positive control (H); no cross-reactivity with non-target viruses was observed;

[0042] Figure 2 A. A low-cost rapid detection method is used to detect the size of sugarcane plants at the jointing stage; B. Samples of the +1 leaf of 20 sugarcane plants; the numbers in the figure represent the corresponding number of each sample; C. RT-PCR test results of the +1 leaf samples of 20 sugarcane plants, where the samples with bands are positive, NTC represents negative, and P represents positive; D. A low-cost rapid detection method for sugarcane yellow leaf virus disease is used to detect the leaves of 20 sugarcane plants at the jointing stage in the field; the numbers in the figure represent the corresponding number of each sample, positive represents the vein imprint of the +1 leaf of a plant with yellow leaf disease, and negative represents the vein imprint of the +1 leaf of a healthy plant; NTC represents negative, and P represents positive. DETAILED DESCRIPTION

[0043] The present invention provides a monoclonal antibody for detecting sugarcane yellow leaf virus. The nucleotide sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID No. 1, and the amino acid sequence is shown as SEQ ID No. 2; the variable region sequence of the heavy chain of the monoclonal antibody is shown as SEQ ID No. 3, and the amino acid sequence is shown as SEQ ID No. 4.

[0044] SEQ ID No. 1:

[0045] ATGATGATCCTGCCCAGTTCCTGTTTCTGCTAGTGCTCTCGATTCAGGAAATCAACGGTGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCTTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATACTAATGGAAAAACCTATTTGAATTGGTTATTACTGAGGCCAGG CCAGTCTCCAAAACGCCTGATCTATCTGGTGTCTAAATTGGACTCTGGAGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTACTGCTTGCAGAGTACACATTTGTATACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA;

[0046] SEQ ID No. 2:

[0047] MMSPAQFLFLLVLSIQEINGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYT NGKTYLNWLLLRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEA EDLGIYYCLQSTHLYTFGGGTKLEIK;

[0048] SEQ ID No.3:

[0049] ATGGGGTTGGAGCTGTATCATCTTCTTTCTGGTAGCAACAGCTACAGGTGTGCACTCCCAGGTCCAGCTGCAGCAGTCTGGGCCTGAGGTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCCGGCTACACATTCACTGATTATGCTATGCACTGGGTGAAGCAGAGTCATGCAAAGAGTCTAGAGTGGATTGGA GTTATTAGTACTTACAATGGTAATACAAACTACAACCAGAAGTTTAAGGGCAAGGCCACAATGACTGTAGACAAATCCTCCAGCACAGCCTATTTGGAACTTGCCAGATTGACATCTGAGGATTCTGCCATCTATTACTGTACAAGAGATGGTTACTACGGAATGGGCTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;

[0050] SEQ ID No.4:

[0051] MGWSCIIFFLVATATGVHSQVQLQQSGPEVVRPGVSVKISCKGSGYTFTD YAMHWVKQSHAKSLEWIGVISTYNGNTNYNQKFKGKATMTVDKSSSTAYLE LARLTSEDSAIYYCTRDGYYGMGYWGQGTSVTVSS.

[0052] In the present invention, the monoclonal antibody is prepared by immunizing an animal with the CP protein encoded by the CP gene. The nucleotide sequence of the CP gene is shown in SEQ ID No. 5, which is as follows:

[0053] SEQ ID No.5:

[0054] .

[0055] The present invention also provides the use of the monoclonal antibody described in the above technical solution in detecting sugarcane yellow leaf virus.

[0056] A method for detecting sugarcane yellow leaf virus, comprising the following steps:

[0057] 1) Take the basal vein of a sugarcane leaf, cut it into a flat surface, and press the cut surface onto a nitrocellulose membrane to obtain a nitrocellulose membrane with an imprinted spot;

[0058] 2) placing the nitrocellulose membrane with the blot obtained in step 1) into a TBST buffer solution containing 2% by weight skim milk for blocking to obtain a blocked nitrocellulose membrane;

[0059] 3) Immersing the blocked nitrocellulose membrane obtained in step 2) in TBST buffer containing 1% by weight skim milk, and then mixing with the monoclonal antibody described in the above technical solution for primary antibody incubation;

[0060] 4) discarding the liquid and washing with TBST buffer to obtain a washed nitrocellulose membrane;

[0061] 5) Immersing the washed nitrocellulose membrane obtained in step 4) in TBST buffer containing 1% by weight skim milk powder, and then mixing with AP-labeled rabbit anti-human IgG antibody for secondary antibody incubation;

[0062] 6) discarding the liquid and washing with TBST buffer to obtain a washed nitrocellulose membrane;

[0063] 7) The washed nitrocellulose membrane obtained in step 6) is mixed with BCIP / NBT color developing solution, and color is developed for 30-35 minutes. The reaction is terminated. When the blot on the nitrocellulose membrane turns purple, the sugarcane leaf is infected with sugarcane yellow leaf virus; when the blot on the nitrocellulose membrane does not turn purple, the sugarcane leaf is not infected with sugarcane yellow leaf virus. The volume ratio of the blot on the washed nitrocellulose membrane to the BCIP / NBT color developing solution is 1:100 μL.

[0064] The present invention involves cutting the basal veins of a sugarcane leaf into a flat surface, and then pressing the surface onto a nitrocellulose membrane to produce a nitrocellulose membrane with an imprinted spot. In the present invention, the pressing time is preferably 10 to 15 seconds. In the present invention, the imprinted spot on the nitrocellulose membrane with an imprinted spot is preferably air-dried before sealing. In the present invention, the nitrocellulose membrane preferably has a size of 6 cm long x 5 cm wide, with a pore size of 0.45 μm (one nitrocellulose membrane can imprint 50 spots).

[0065] The present invention blocks the obtained nitrocellulose membrane with the blotted spot by placing it in a TBST buffer solution containing 2% skim milk by weight, thereby obtaining a blocked nitrocellulose membrane. In the present invention, the volume ratio of the blotted nitrocellulose membrane to the TBST buffer solution is preferably 1:35 mL. In the present invention, the blocking conditions preferably include blocking at 20-30°C for 1 hour. In the present invention, the blotted nitrocellulose membrane is preferably placed in a box measuring 9.0 cm (length) × 6.0 cm (width) × 3.5 cm (height) for blocking.

[0066] The present invention immerses the obtained blocked nitrocellulose membrane in a TBST buffer solution containing 1% skim milk by mass, and then mixes it with the monoclonal antibody described in the above technical solution for primary antibody incubation. In the present invention, the monoclonal antibody is mixed in the form of a monoclonal antibody solution at a concentration of 2.7 mg / mL. In the present invention, the number of the blocked nitrocellulose membranes, the volume of the TBST buffer solution, and the volume ratio of the monoclonal antibody solution are preferably 1:35 mL:4.6 μL. In the present invention, the conditions for the primary antibody incubation preferably include: incubation at a temperature of 20-30°C for 3 hours and a rotation speed of 15 rpm.

[0067] The present invention discards the liquid and uses TBST buffer to wash the membrane to obtain a washed nitrocellulose membrane. In the present invention, the washing conditions preferably include: a rotation speed of 40 rpm, two washes, each wash for 5 minutes.

[0068] The present invention immerses the washed nitrocellulose membrane in a TBST buffer solution containing 1% skim milk powder by mass, and then mixes it with an AP-labeled rabbit anti-human IgG antibody for secondary antibody incubation. In the present invention, the AP-labeled rabbit anti-human IgG antibody is preferably mixed in the form of an AP-labeled rabbit anti-human IgG antibody solution with a concentration of 0.2 mg / ml. In the present invention, the ratio of the number of washed nitrocellulose membranes to the volume of the TBST buffer solution and the volume of the AP-labeled rabbit anti-human IgG antibody solution is preferably 1:35 mL:10 μL. In the present invention, the conditions for the secondary antibody incubation preferably include: incubation overnight at 4°C and a rotation speed of 15 rpm.

[0069] The present invention discards the liquid and uses TBST buffer to wash the membrane to obtain a washed nitrocellulose membrane. In the present invention, the washing conditions preferably include: a rotation speed of 40 rpm, two washes, each time for 5 minutes.

[0070] The present invention mixes the washed nitrocellulose membrane with a BCIP / NBT color developing solution, develops color for 30 to 35 minutes, and terminates the reaction. When the blot on the nitrocellulose membrane turns purple, the sugarcane leaf is infected with sugarcane yellow leaf virus; when the blot on the nitrocellulose membrane does not develop color, the sugarcane leaf is not infected with sugarcane yellow leaf virus. The volume ratio of the number of blots on the washed nitrocellulose membrane to the BCIP / NBT color developing solution is 1:100 μL. Preferably, the developed nitrocellulose membrane is placed in water to terminate the reaction.

[0071] The main reagents, consumables and instruments in the present invention are as follows:

[0072] Nitrocellulose membrane: Merck, product number: HATF00010.

[0073] Skim milk: Saiguo Biotechnology Co., Ltd., product number: 1172.

[0074] 10×TBS: White Shark Biotechnology Co., Ltd., product number: BL608A.

[0075] Tween-20: White Shark Biotechnology Co., Ltd., product number: BS100.

[0076] AP-labeled rabbit anti-human IgG antibody: Sangon Biotech (Shanghai) Co., Ltd., catalog number: D110153.

[0077] BCIP / NBT color development kit: Beijing Solebow Technology Co., Ltd., catalog number: PR1100.

[0078] Small rocking chair: American Cylo Czech, item number: SLK-R3000-S.

[0079] Microscope: An ordinary stereo microscope will do.

[0080] Preparation of TBST buffer: Take 100 mL of the above 10×TBS, add 800 mL of water, and then add 500 μL of Tween-20 to make up to 1 L.

[0081] Prepare 100 mL of 2% skim milk TBST buffer by adding 2 g of skim milk to 90 mL of the above TBST buffer and dilute to 100 mL.

[0082] Prepare 100 mL of 1% skim milk TBST buffer by adding 1 g of skim milk to 90 mL of the above TBST buffer and dilute to 100 mL.

[0083] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0084] Example 1

[0085] Preparation of monoclonal antibodies

[0086] 1. Construction of expression vector and protein expression and purification

[0087] The CP gene sequence (SEQ OD No. 3) was downloaded from the NCIB website and optimized using the MaxCodon™ Optimization Program (V13) software. The CP gene was then inserted into the expression vector pET30a using full gene synthesis and restriction enzymes at the NdeI and HindIII sites. The expression vector was transformed into the BL21(DE3) expression strain, the strain was harvested, induced to express the CP protein, and purified.

[0088] 2. Animal immunization

[0089] The amount of antigen required for immunization was diluted with physiological saline, and the adjuvant and antigen were thoroughly mixed at a volume ratio of 1:1.

[0090] The prepared immunogen was injected intraperitoneally into mice, and a small amount of tail blood was collected to determine the serum titer by indirect ELISA. The serum titer of the mice was 1:512K.

[0091] 3. B cell sorting

[0092] Take the mouse spleen, remove the surrounding connective tissue, place the spleen on a cell strainer, and gently press the spleen with a syringe needle to obtain a cell suspension. Take Ficoll-Paque PLUS cell separation solution and cell suspension at a volume ratio of 1:2 and add them to a test tube. Centrifuge at 1500rpm for 15 minutes. The middle layer of the test tube after centrifugation is generally a mononuclear cell layer. Aspirate the mononuclear cell layer and add SOL024 solution. Centrifuge at 1500rpm for 10 minutes. Remove the supernatant after centrifugation and add 500μL SOL024 solution to resuspend the cells. The antigen is fluorescently labeled, and the BCR on the surface of MBC can specifically bind to the antigen. The MBC that specifically binds to the antigen will carry a fluorescent label and a marker on the surface of B cells. The antigen-specific MBC can be sorted out one by one from the cell suspension by flow cytometry and placed in a 96-well plate. And perform High-throughput expression.

[0093] 4. Screening of positive cell supernatants and monoclonal antibody gene sequencing

[0094] Monoclonal antibody cell supernatants were obtained through high-throughput expression, and then detected using indirect ELISA to select positive clones. The light chain variable region sequences (SEQ ID No. 1) and heavy chain variable region sequences (SEQ ID No. 3) were determined, and expression vectors were constructed based on these sequences. Expression and purification were performed to obtain monoclonal antibodies.

[0095] Example 2

[0096] A low-cost, rapid method for detecting and determining sugarcane yellow leaf virus infection

[0097] (a) Leaf vein imprint: Take the basal veins of one leaf of sugarcane with yellow leaf disease and one leaf of healthy sugarcane (such as Figure 1 Middle A), cut it into a flat surface with a sharp blade, and then press the cut surface firmly onto a nitrocellulose membrane for 10-15 seconds to obtain a blot.

[0098] (b) Blocking: After the blot was air-dried, the nitrocellulose membrane was placed in a 9.0 (L) × 6.0 (W) × 3.5 (H) box. 35 mL of TBST buffer containing 2% skim milk was added and the membrane was placed on a small shaker (set to 15 rpm) and blocked at room temperature for 1 h.

[0099] (c) Incubation with primary antibody: The blocked NC membrane was immersed in 35 ml of TBST buffer containing 1% skim milk powder, and 4.6 μL of 2.7 mg / mL monoclonal antibody (Example 1) was added. The membrane was placed on a small shaker (set to 15 rpm) and incubated at room temperature for 3 h.

[0100] (d) Washing: Discard the incubation solution, add 40 mL of TBST buffer, place on a small shaker (set to 40 rpm) and wash for 5 min. Repeat the washing twice.

[0101] (e) Incubation with secondary antibody: Immerse the washed NC membrane in 35 mL of 1% skim milk powder in TBST buffer, add 10 μL of AP-labeled rabbit anti-human IgG antibody (concentration: 0.2 mg / mL), place on a small shaker (set to 15 rpm), and incubate at 4°C overnight.

[0102] (f) Washing: Pour off the incubation solution, add 40 mL of TBST buffer, place on a small shaker (set to 40 rpm) and wash for 5 min. Repeat the washing twice.

[0103] (e) Add substrate for color development: Pour off the wash solution and add 100 μL of BCIP / NBT color development solution per blot on the NC membrane. Develop for 30-35 min. When the positive control blot shows a purple spot, place the membrane in water to terminate the reaction.

[0104] (g) Result identification: Observe with the naked eye or microscope. The tissue spots of the sample infected with sugarcane yellow leaf virus will be purple, while the tissue spots of the uninfected sample will not show any color. (Results are shown in the figure below.) Figure 1 Middle B).

[0105] Example 3

[0106] Sensitivity assessment

[0107] (a) To evaluate the sensitivity of the monoclonal antibodies prepared in the present invention, the purified CP protein (initial concentration: 0.6 mg / mL) was diluted 10-fold into seven gradients. Each gradient (5.0 μL) was spotted onto a NC membrane and air-dried.

[0108] (b) Blocking: After the blots were air-dried, the nitrocellulose membrane (containing 7 blots) was placed in a 9.0 (L) × 6.0 (W) × 3.5 (H) box. 35 mL of TBST buffer containing 2% skim milk was added and the membrane was placed on a small shaker (set to 15 rpm) and blocked at room temperature for 1 h.

[0109] (c) Incubation with primary antibody: The blocked NC membrane was immersed in 35 ml of TBST buffer containing 1% skim milk powder, and 4.6 μL of 2.7 mg / mL monoclonal antibody (Example 1) was added. The membrane was placed on a small shaker (set to 15 rpm) and incubated at room temperature for 3 h.

[0110] (d) Washing: Discard the incubation solution, add 40 mL of TBST buffer, place on a small shaker (set to 40 rpm) and wash for 5 min. Repeat the washing twice.

[0111] (e) Incubation with secondary antibody: Immerse the washed NC membrane in 35 mL of 1% skim milk powder in TBST buffer, add 10 μL of (AP)-labeled rabbit anti-human IgG antibody (concentration: 0.2 mg / mL), place on a small shaker (set to 15 rpm), and incubate at 4°C overnight.

[0112] (f) Washing: Pour off the incubation solution, add 40 mL of TBST buffer, place on a small shaker (set to 40 rpm) and wash for 5 min. Repeat the washing twice.

[0113] (e) Add substrate for color development: Discard the wash solution and add 35 ml of BCIP / NBT color development solution (100 μL per blot spot) to the NC membrane. Develop for 30-35 min. When the positive control blot shows a purple spot, place the membrane in water to terminate the reaction.

[0114] (g) Result interpretation: With naked eye observation, faint but discernible spots were observed at a concentration of 30 pg, while no visible signal was detected at a concentration of 3 pg (results as shown in Figure 1 Middle C).

[0115] Example 4

[0116] Specificity assessment

[0117] (a) The midribs of three leaves infected with sugarcane mosaic virus (SCMV), sorghum mosaic virus (SrMV), and sugarcane streak mosaic virus (SCSMV) were blotted (prepared as in Example 2).

[0118] (b) Blocking: After the blot was air-dried, the nitrocellulose membrane was placed in a 9.0 (L) × 6.0 (W) × 3.5 (H) box. 35 mL of TBST buffer containing 2% skim milk was added and the membrane was placed on a small shaker (set to 15 rpm) and blocked at room temperature for 1 h.

[0119] (c) Incubation with primary antibody: The blocked NC membrane was immersed in 35 ml of TBST buffer containing 1% skim milk powder, and 4.6 μL of 2.7 mg / mL monoclonal antibody (Example 1) was added. The membrane was placed on a small shaker (set to 15 rpm) and incubated at room temperature for 3 h.

[0120] (d) Washing: Pour off the incubation solution, add 40 mL of TBST buffer, place on a small shaker (set to 40 rpm) and wash for 5 min. Repeat the washing twice.

[0121] (e) Incubation with secondary antibody: Immerse the washed NC membrane in 35 mL of 1% skim milk powder in TBST buffer, add 10 μL of (AP)-labeled rabbit anti-human IgG antibody (concentration: 0.2 mg / mL), place on a small shaker (speed set to 15 rpm), and incubate at 4°C overnight.

[0122] (f) Washing: Pour off the incubation solution, add 40 mL of TBST buffer, place on a small shaker (set to 40 rpm) and wash for 5 min. Repeat the washing twice.

[0123] (e) Add substrate for color development: Discard the wash solution and add 100 μL of BCIP / NBT color development solution per blot on the NC membrane. Develop for 30-35 min. When the positive control blot shows a purple spot, place the membrane in water to terminate the reaction.

[0124] (g) Result evaluation: Observe with the naked eye or microscope. Purple spots appear in the positive control, while no purple spots are found in the negative control and the blots infected with the three viruses (results are shown in Figure 2). Figure 1 (DH).

[0125] Example 5

[0126] A low-cost and rapid method for detecting sugarcane yellow leaf virus disease is developed to detect sugarcane leaves at the jointing stage in the field.

[0127] The low-cost rapid detection method for sugarcane yellow leaf virus disease was established to detect asymptomatic sugarcane leaves at the jointing stage in the field. The size of sugarcane at the jointing stage (such as Figure 2As shown in Figure 2), 20 sugarcane plants were selected for testing, of which FN14-307, XTT22, FN14-128, FN14-400, FN14-83, FN14-71, FN14-98, FN14-325, FN14-398, FN14-23, and FN14-213 were negative samples, and FN14-276, FN14-85, FN14-151, FN14-209, FN14-13, FN14-25, FN14-228, YT93-159, and FN14-171 were positive samples. +1 leaf sample (as shown in Figure 2) Figure 2 From Figure 2 The results showed that the newly established detection method could detect samples infected with sugarcane yellow leaf virus from field samples during the jointing stage of sugarcane.

[0128] The detection steps are as follows:

[0129] (a) Leaf vein imprint: The basal veins of one leaf of sugarcane infected with yellow leaf disease and one leaf of healthy sugarcane were cut into planes with a sharp blade. The cut surface was then pressed firmly onto a nitrocellulose membrane for 10-15 seconds to obtain an imprint spot.

[0130] (b) Blocking: After the blot was air-dried, the nitrocellulose membrane was placed in a 9.0 (L) × 6.0 (W) × 3.5 (H) box. 35 mL of TBST buffer containing 2% skim milk was added and the membrane was placed on a small shaker (set to 15 rpm) and blocked at room temperature for 1 h.

[0131] (c) Incubation with primary antibody: The blocked NC membrane was immersed in 35 ml of TBST buffer containing 1% skim milk powder, and 4.6 μL of 2.7 mg / mL monoclonal antibody (Example 1) was added. The membrane was placed on a small shaker (set to 15 rpm) and incubated at room temperature for 3 h.

[0132] (d) Washing: Discard the incubation solution, add 40 mL of TBST buffer, place on a small shaker (set to 40 rpm) and wash for 5 min. Repeat the washing twice.

[0133] (e) Incubation with secondary antibody: Immerse the washed NC membrane in 35 mL of 1% skim milk powder in TBST buffer, add 10 μL of (AP) labeled rabbit anti-human IgG antibody (concentration: 0.2 mg / mL), place on a small shaker (speed set to 15 rpm), and incubate at 4°C overnight.

[0134] (f) Washing: Pour off the incubation solution, add 40 mL of TBST buffer, place on a small shaker (set to 40 rpm) and wash for 5 min. Repeat the washing twice.

[0135] (e) Add substrate for color development: Discard the wash solution and add 100 μL of BCIP / NBT color development solution per blot on the NC membrane. Develop for 30-35 min. When the positive control blot shows a purple spot, place the membrane in water to terminate the reaction.

[0136] (g) Result identification: Observe with the naked eye or microscope. The tissue spots of the sample infected with sugarcane yellow leaf virus will appear purple, while the tissue spots of the uninfected sample will not show any color.

[0137] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A monoclonal antibody for detecting sugarcane yellow leaf virus, characterized in that: The nucleotide sequence of the variable region of the light chain of the monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2; the variable region sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No.

4.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody is prepared by immunizing an animal with the CP protein encoded by the CP gene.

3. The monoclonal antibody according to claim 1, characterized in that The nucleotide sequence of the CP gene is shown in SEQ ID No.

5.

4. Use of the monoclonal antibody according to any one of claims 1 to 3 in detecting sugarcane yellow leaf virus.

5. A method for detecting sugarcane yellow leaf virus, characterized in that: The following steps are involved: 1) Take the base vein of a sugarcane leaf, cut it into a flat surface, and press the cut surface onto a nitrocellulose membrane to obtain a nitrocellulose membrane with an imprinted spot; 2) placing the nitrocellulose membrane with the blot obtained in step 1) into a TBST buffer solution containing 2% by weight skim milk for blocking to obtain a blocked nitrocellulose membrane; 3) immersing the blocked nitrocellulose membrane obtained in step 2) in a TBST buffer solution containing 1% by weight skim milk, and then mixing with the monoclonal antibody according to any one of claims 1 to 3, and incubating with the primary antibody; 4) discarding the liquid and washing with TBST buffer to obtain a washed nitrocellulose membrane; 5) Immersing the washed nitrocellulose membrane obtained in step 4) in TBST buffer containing 1% by weight skim milk powder, and then mixing with AP-labeled rabbit anti-human IgG antibody for secondary antibody incubation; 6) discarding the liquid and washing with TBST buffer to obtain a washed nitrocellulose membrane; 7) mixing the washed nitrocellulose membrane obtained in step 6) with a BCIP / NBT color developing solution, developing color for 30-35 minutes, and terminating the reaction. When the blot on the nitrocellulose membrane turns purple, the sugarcane leaves are infected with sugarcane yellow leaf virus; when the blot on the nitrocellulose membrane does not turn purple, the sugarcane leaves are not infected with sugarcane yellow leaf virus. The volume ratio of the number of blots contained in the washed nitrocellulose membrane to the volume of BCIP / NBT color development solution was 1:100 μL.

6. The method according to claim 5, characterized in that The pressing time in step 1) is 10 to 15 seconds; The blot on the nitrocellulose membrane with the blot is air-dried and then blocked; The specifications of the nitrocellulose membrane are 6 cm in length and 5 cm in width, and the pore size is 0.45 μm.

7. The method according to claim 5, characterized in that In step 2), the volume ratio of the nitrocellulose membrane with the blotted spot to the TBST buffer is 1:35 mL; The blocking conditions include: blocking at 20-30° C. for 1 hour.

8. The method according to claim 5, characterized in that In step 3), the monoclonal antibodies are mixed in the form of a monoclonal antibody solution at a concentration of 2.7 mg / mL; The volume ratio of the blocked nitrocellulose membrane, the volume of TBST buffer, and the monoclonal antibody solution was 1:35 mL:4.6 μL; The primary antibody incubation conditions include: incubation at a temperature of 20-30° C. for 3 hours and a rotation speed of 15 rpm.

9. The method according to claim 5, characterized in that The washing conditions in steps 4) and 6) both include: a rotation speed of 40 rpm, two washes, each for 5 minutes.

10. The method according to claim 5, characterized in that In step 5), AP-labeled rabbit anti-human IgG antibody is mixed in the form of an AP-labeled rabbit anti-human IgG antibody solution at a concentration of 0.2 mg / ml; The volume ratio of the washed nitrocellulose membrane to the volume of TBST buffer and AP-labeled rabbit anti-human IgG antibody solution is 1:35 mL:10 μL; The secondary antibody incubation conditions include: incubation at 4° C. overnight and a rotation speed of 15 rpm.

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