A rapid test strip for citrus huanglongbing bacterium nucleic acid colloidal gold and a preparation method thereof
Patent Information
- Application Number
- CN202610848345.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-12
- Publication Date
- 2026-09-08
AI Technical Summary
然而，目前针对柑橘黄龙病菌的胶体金检测试纸条大多基于抗原 - 抗体反应，存在抗体制备难度大、批次间差异大、灵敏度不足等问题
[0032] High sensitivity: This invention uses asymmetric PCR to amplify the target nucleic acid sequence, combined with nucleic acid hybridization and colloidal gold immunochromatography, achieving a detection sensitivity of up to 10 copies/μL, which is far higher than traditional serological detection methods.
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Figure CN122706489A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular detection technology for plant diseases, specifically relating to a rapid test strip for detecting Huanglongbing (HLB) of citrus based on nucleic acid hybridization and colloidal gold immunochromatography, as well as the preparation method and usage method of the test strip. Background Technology
[0002] Huanglongbing (HLB) is a devastating disease of citrus caused by bacteria of the genus *Candidatus Liberibacter*. It is primarily spread through the citrus psyllid and grafting. This disease is widespread in major citrus-producing regions worldwide, causing tree vigor decline, reduced fruit quality, and a sharp decrease in yield. In severe cases, it can lead to the death of the entire tree, resulting in enormous economic losses to the citrus industry.
[0003] Currently, the main methods for detecting citrus Huanglongbing (HLB) include symptom observation, indicator plant grafting, serological testing, and molecular biological testing. Symptom observation is highly subjective and prone to missed diagnoses when early symptoms are not obvious; indicator plant grafting is time-consuming, requiring several months to obtain results; serological testing has low sensitivity and is prone to false positives; molecular biological testing methods, such as polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR), have the advantages of high sensitivity and specificity, but require specialized equipment and technicians, and the testing cycle is long, making them unsuitable for rapid on-site testing in the field.
[0004] Colloidal gold immunochromatography is a rapid detection technique that combines immunoassay and chromatographic analysis. It offers advantages such as ease of operation, speed, intuitive results, and the elimination of the need for expensive equipment, and has been widely applied in medicine, food safety, and agriculture. However, most current colloidal gold test strips for citrus Huanglongbing (HLB) are based on antigen-antibody reactions, which suffer from challenges such as difficult antibody preparation, significant batch-to-batch variability, and insufficient sensitivity. Colloidal gold test strips based on nucleic acid hybridization can overcome these shortcomings by enabling highly sensitive and specific detection of HLB through the binding of specific nucleic acid probes to target nucleic acid sequences.
[0005] Therefore, developing a simple, rapid, sensitive, specific, and field-ready colloidal gold rapid test strip for the nucleic acid of citrus Huanglongbing (HLB) and its preparation method is of great significance for the early diagnosis and control of HLB. Summary of the Invention
[0006] (a) Technical problems to be solved
[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a rapid nucleic acid colloidal gold test strip for citrus Huanglongbing (HLB) and its preparation method. This test strip is based on nucleic acid hybridization and colloidal gold immunochromatography technology, which can rapidly, sensitively and specifically detect HLB in citrus. It is easy to operate, provides intuitive results, does not require expensive instruments and equipment, and is suitable for rapid on-site detection in the field.
[0008] (II) Technical Solution
[0009] To achieve the above objectives, the present invention adopts the following technical solution:
[0010] A rapid colloidal gold test strip for nucleic acid detection of Huanglongbing (HLB) of citrus includes a PVC base plate, and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially pasted onto the PVC base plate; the conjugate pad is coated with a colloidal gold-labeled detection probe; the nitrocellulose membrane is sequentially coated with a detection line (T line) and a control line (C line); the detection line is coated with a capture probe, and the control line is coated with streptavidin.
[0011] Furthermore, the sequence of the detection probe is 5'-biotin-ATGCGTATTTGCGGCGGATG-3' (SEQ ID NO:1), and the sequence of the capture probe is 5'-NH2-TTTTTTTTTTCCGCAATCCCTGACGCTACC-3' (SEQ ID NO:2).
[0012] Furthermore, the colloidal gold particles have a particle size of 15-25 nm, preferably 20 nm.
[0013] Furthermore, the sample pad is a glass fiber membrane, the bonding pad is a glass fiber membrane or a polyester membrane, and the absorbent pad is absorbent filter paper.
[0014] The preparation method of the above-mentioned rapid nucleic acid colloidal gold test strip for Huanglongbing bacteria of citrus includes the following steps:
[0015] Preparation of colloidal gold solution: Colloidal gold solution with a particle size of 15-25 nm was prepared by the trisodium citrate reduction method;
[0016] Preparation of colloidal gold labeled detection probe: The detection probe was mixed with colloidal gold solution, a stabilizer was added for labeling, and after centrifugation purification, it was resuspended in the labeling preservation solution to obtain the colloidal gold labeled detection probe.
[0017] Preparation of the conjugate pad: The colloidal gold-labeled detection probe is uniformly sprayed onto the conjugate pad and dried under vacuum for later use;
[0018] Treatment of nitrocellulose membrane: The capture probe and streptavidin are sprayed onto the nitrocellulose membrane to form the detection line and the quality control line, respectively. After vacuum drying, it is ready for use.
[0019] Assembly of test strips: Attach the sample pad, the prepared conjugate pad, the nitrocellulose membrane, and the absorbent pad to the PVC base plate in sequence, cut into test strips with a width of 3-5mm, and seal for storage.
[0020] Further, the preparation method of the colloidal gold solution in step 1 is as follows: heat 100 mL of 0.01% chloroauric acid solution to boiling, quickly add 1.5-2.5 mL of 1% trisodium citrate solution, continue heating and boiling for 10-15 min until the solution turns wine red, stop heating, cool to room temperature, make up to 100 mL with ultrapure water, and store at 4°C protected from light.
[0021] Further, the preparation method of the colloidal gold-labeled detection probe in step 2 is as follows: Take 10 mL of colloidal gold solution, adjust the pH to 8.0-8.5 with 0.2 M K2CO3 solution, add 10-20 μL of 100 μM detection probe, and gently shake and incubate at room temperature for 30-60 min; add 1 mL of 10% BSA solution, and block at room temperature for 30 min; centrifuge at 12000 rpm for 15 min, discard the supernatant, resuspend the precipitate in labeling preservation solution to 1 mL, and store at 4℃ in the dark.
[0022] Furthermore, the composition of the labeling preservation solution is: 20mM Tris-HCl (pH 8.0), 1% BSA, 0.5% Tween-20, 5% sucrose, and 0.02% NaN3.
[0023] Further, the preparation method of the conjugate pad in step 3 is as follows: the colloidal gold-labeled detection probe is uniformly sprayed onto the conjugate pad at a rate of 4-6 μL / cm using a gold sprayer, vacuum dried at 37°C for 2-4 hours, sealed and stored in a desiccator.
[0024] Further, the specific method for treating the nitrocellulose membrane in step 4 is as follows: 100 μM capture probe and 1 mg / mL streptavidin are sprayed onto the nitrocellulose membrane at a rate of 1-2 μL / cm using a spotting apparatus. The distance between the detection line and the control line is 4-6 mm. The membrane is then vacuum dried at 37°C for 2-4 hours, sealed, and stored in a desiccator.
[0025] The method of using the above-mentioned rapid nucleic acid colloidal gold test strip for citrus Huanglongbing bacteria includes the following steps:
[0026] Sample processing: Take midrib tissue from citrus leaves, grind it, add nucleic acid extraction solution, and extract total nucleic acid;
[0027] Amplification reaction: The 16S rRNA gene fragment of Huanglongbing fungus was amplified by asymmetric PCR to obtain single-stranded DNA product;
[0028] Detection: Add the amplification product to the sample pad of the test strip, react at room temperature for 10-15 minutes, and then observe the results;
[0029] Result interpretation: If both the test line and the control line show red bands, it indicates that the sample contains citrus Huanglongbing fungus; if only the control line shows a red band, it indicates that the sample does not contain citrus Huanglongbing fungus; if the control line does not show a red band, it indicates that the test strip is invalid.
[0030] (III) Beneficial Effects
[0031] Compared with the prior art, the present invention has the following advantages:
[0032] High sensitivity: This invention uses asymmetric PCR to amplify the target nucleic acid sequence, combined with nucleic acid hybridization and colloidal gold immunochromatography, achieving a detection sensitivity of up to 10 copies / μL, which is far higher than traditional serological detection methods.
[0033] High specificity: The detection probe and capture probe designed in this invention target the conserved region of the 16S rRNA gene of Huanglongbing fungus in citrus, and have no cross-reaction with other citrus pathogens, thus exhibiting high specificity.
[0034] Fast and simple: The entire testing process (including sample processing, amplification and detection) takes only 2-3 hours. It is easy to operate, does not require professional instruments and equipment or technicians, and the results are intuitive and visible, making it suitable for rapid on-site testing in the field.
[0035] Good stability: The test strips prepared by this invention can be stored for more than 12 months at 4℃, showing good stability and making them easy to transport and store. Attached Figure Description
[0036] To provide a more detailed description of the technical solutions of the embodiments of this invention, the accompanying drawings used in the following description of the embodiments are briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on such drawings without creative effort.
[0037] Figure 1 This is a schematic diagram of the overall structure of the rapid nucleic acid colloidal gold test strip for citrus Huanglongbing bacteria described in this invention;
[0038] Figure 2This is a schematic diagram illustrating the determination of the detection results of the rapid nucleic acid colloidal gold test strip for Huanglongbing bacteria in citrus described in this invention. Detailed Implementation
[0039] The present invention will be further described in detail below with reference to specific embodiments.
[0040] Example 1
[0041] Preparation of colloidal gold solution
[0042] 100 mL of 0.01% chloroauric acid solution was added to a 250 mL round-bottom flask and heated to boiling. Then, 2.0 mL of 1% trisodium citrate solution was quickly added, and the mixture was heated and boiled for another 12 minutes until the solution turned a uniform wine-red color. Heating was then stopped, and the solution was cooled to room temperature. The volume was then adjusted to 100 mL with ultrapure water and stored at 4°C protected from light. The particle size of the colloidal gold particles was observed using a transmission electron microscope. The results showed that the colloidal gold particles were uniform in size, with an average particle size of approximately 20 nm.
[0043] Preparation of colloidal gold labeled detection probes
[0044] Take 10 mL of the colloidal gold solution prepared above, adjust the pH to 8.2 with 0.2 M K2CO3 solution, add 15 μL of 100 μM detection probe (5'-biotin-ATGCGTATTTGCGGCGGATG-3'), and incubate gently with shaking at room temperature for 45 min. Add 1 mL of 10% BSA solution and block at room temperature for 30 min. Centrifuge at 12000 rpm for 15 min, discard the supernatant, and resuspend the precipitate in labeled preservation solution (20 mM Tris-HCl pH 8.0, 1% BSA, 0.5% Tween-20, 5% sucrose, 0.02% NaN3) to 1 mL, and store at 4 °C protected from light.
[0045] Preparation of binding pad
[0046] Glass fiber membranes were cut into 2cm × 30cm strips and soaked in a treatment solution (20mM Tris-HCl pH 8.0, 0.5% Tween-20, 5% sucrose) for 30 min, then dried at 37℃. Colloidal gold-labeled detection probes were uniformly sprayed onto the treated glass fiber membranes at a rate of 5 μL / cm using a gold sputtering apparatus. The membranes were then vacuum-dried at 37℃ for 3 h, sealed, and stored in a desiccator.
[0047] Treatment of nitrocellulose membranes
[0048] Nitrocellulose membranes were cut into 3cm × 30cm strips and adhered to a PVC substrate. A 100μM capture probe (5'-NH2-TTTTTTTTTTCCGCAATCCCTGACGCTACC-3') and 1mg / mL streptavidin were sprayed onto the nitrocellulose membranes at a rate of 1.5μL / cm using a spotting apparatus. The distance between the detection line and the control line was 5mm. The membranes were vacuum dried at 37℃ for 3 hours, sealed, and stored in a desiccator.
[0049] Assembly of test strips
[0050] The sample pad (glass fiber membrane), the treated conjugate pad, the nitrocellulose membrane, and the absorbent pad (absorbent filter paper) are sequentially attached to the PVC base plate, with each part overlapping by 1-2 mm. The assembled plate is then cut into 4 mm wide test strips using a strip cutter, which are placed in plastic cartridges, sealed, and stored at 4°C.
[0051] Example 2
[0052] The recombinant plasmid containing the 16S rRNA gene fragment of *Citrus citrus Huanglongbing* was serially diluted to concentrations of 10^6, 10^5, 10^4, 10^3, 10^2, 10^1, and 10^0 copies / μL. Asymmetric PCR was used for amplification. The amplification system consisted of 25 μL: 12.5 μL of 2×PCR Mix, 0.5 μL of upstream primer (10 μM), 2.5 μL of downstream primer (10 μM), 2 μL of template DNA, and 7.5 μL of ultrapure water. The amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 10 min.
[0053] 10 μL of amplification product was added to the sample pad of the test strip, and the reaction was carried out at room temperature for 15 min before the results were observed. The results showed that when the template concentration was 10^1 copies / μL, a clear red band was still visible in the detection line, indicating that the detection sensitivity of the test strip of this invention can reach 10 copies / μL.
[0054] Example 3
[0055] Total nucleic acid was extracted from citrus plants infected with Huanglongbing (HLB), citrus canker, and anthracnose, as well as healthy citrus plants, and detected using the test strip of this invention. The results showed that only samples from citrus plants infected with HLB showed red bands on both the test line and the control line, while other samples only showed red bands on the control line. This indicates that the test strip of this invention does not cross-react with other citrus pathogens and has good specificity.
[0056] Example 4
[0057] The prepared test strips were stored at 4℃, 25℃, and 37℃, respectively. A portion of the test strips was taken out every month and detected using 10^3 copies / μL of recombinant plasmid amplification product. The results showed that the test strips stored at 4℃ for 12 months exhibited no significant color change in the detection line and control line, and the detection sensitivity remained at 10 copies / μL. The test strips stored at 25℃ for 6 months showed no significant decrease in detection performance. The test strips stored at 37℃ for 3 months maintained good detection performance. This indicates that the test strips of the present invention have good stability.
[0058] Example 5
[0059] One hundred leaf samples suspected of being infected with citrus Huanglongbing (HLB) were collected from citrus-producing areas and tested using the test strips of this invention and real-time quantitative PCR. The results showed that the test strips of this invention detected 32 positive samples and 68 negative samples; the real-time quantitative PCR method detected 33 positive samples and 67 negative samples. The concordance rate between the two methods was 99%, indicating that the test strips of this invention can be used for rapid detection of field samples.
Claims
1. A rapid nucleic acid colloidal gold test strip for citrus Huanglongbing bacteria, characterized in that, The device includes a PVC base plate, and a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially attached to the PVC base plate; the conjugate pad is coated with a colloidal gold-labeled detection probe; the nitrocellulose membrane is sequentially coated with a detection line and a control line; the detection line is coated with a capture probe, and the control line is coated with streptavidin.
2. The rapid nucleic acid colloidal gold test strip for citrus Huanglongbing bacteria according to claim 1, characterized in that, The sequence of the detection probe is SEQ ID NO:1, and the sequence of the capture probe is SEQ ID NO:
2.
3. The rapid nucleic acid colloidal gold test strip for citrus Huanglongbing bacteria according to claim 1, characterized in that, The colloidal gold particles have a particle size of 15-25 nm.
4. The rapid nucleic acid colloidal gold test strip for citrus Huanglongbing bacteria according to claim 1, characterized in that, The sample pad is a glass fiber membrane, the bonding pad is a glass fiber membrane or a polyester membrane, and the absorbent pad is absorbent filter paper.
5. A method for preparing a rapid nucleic acid colloidal gold test strip for *Citrus citrus Huanglongbing* as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) A colloidal gold solution with a particle size of 15-25 nm was prepared by the trisodium citrate reduction method; (2) The detection probe was mixed with colloidal gold solution, a stabilizer was added for labeling, and after centrifugation and purification, it was resuspended in the labeling preservation solution to obtain the colloidal gold labeled detection probe. (3) Spray the colloidal gold-labeled detection probe evenly onto the conjugate pad, and dry it under vacuum for later use; (4) Spray the capture probe and streptavidin onto the nitrocellulose membrane to form the detection line and the quality control line, and then dry them under vacuum for later use. (5) Paste the sample pad, the prepared binding pad, the nitrocellulose membrane and the absorbent pad onto the PVC base plate in sequence, cut them into test strips with a width of 3-5mm, and seal them for storage.
6. The preparation method according to claim 5, characterized in that, The specific method for preparing the colloidal gold solution in step (1) is as follows: Heat 100 mL of 0.01% chloroauric acid solution to boiling, quickly add 1.5-2.5 mL of 1% trisodium citrate solution, continue heating and boiling for 10-15 min until the solution turns wine red, stop heating, cool to room temperature, make up to 100 mL with ultrapure water, and store at 4°C protected from light.
7. The preparation method according to claim 5, characterized in that, The specific preparation method of the colloidal gold labeled detection probe in step (2) is as follows: Take 10 mL of colloidal gold solution, adjust the pH to 8.0-8.5 with 0.2 M K2CO3 solution, add 10-20 μL of 100 μM detection probe, and gently shake and incubate at room temperature for 30-60 min; add 1 mL of 10% BSA solution, and block at room temperature for 30 min; centrifuge at 12000 rpm for 15 min, discard the supernatant, resuspend the precipitate in label preservation solution to 1 mL, and store at 4℃ in the dark.
8. The preparation method according to claim 7, characterized in that, The labeling and preservation solution consists of: 20 mM Tris-HCl (pH 8.0), 1% BSA, 0.5% Tween-20, 5% sucrose, and 0.02% NaN3.
9. The preparation method according to claim 5, characterized in that, The specific method for preparing the conjugate pad in step (3) is as follows: the colloidal gold-labeled detection probe is uniformly sprayed onto the conjugate pad at a rate of 4-6 μL / cm using a gold sprayer, vacuum dried at 37℃ for 2-4 h, sealed and stored in a desiccator; the specific method for treating the nitrocellulose membrane in step (4) is as follows: 100 μM capture probe and 1 mg / mL streptavidin are respectively sprayed onto the nitrocellulose membrane at a rate of 1-2 μL / cm using a spotting apparatus, the distance between the detection line and the control line is 4-6 mm, vacuum dried at 37℃ for 2-4 h, sealed and stored in a desiccator.
10. A method of using a rapid nucleic acid colloidal gold test strip for citrus Huanglongbing bacteria as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Take the midrib tissue of citrus leaves, grind it, add nucleic acid extraction solution, and extract total nucleic acid; (2) The 16S rRNA gene fragment of Huanglongbing fungus was amplified by asymmetric PCR to obtain a single-stranded DNA product; (3) Add the amplification product to the sample pad of the test strip, react at room temperature for 10-15 minutes and then observe the results; (4) If both the test line and the control line show red bands, it indicates that the sample contains citrus Huanglongbing fungus; if only the control line shows red bands, it indicates that the sample does not contain citrus Huanglongbing fungus. If no red band appears on the control line, the test strip is invalid.