Primer and probe composition for detecting chrysanthemum B virus, kit and detection method and application thereof

Abstract

The invention discloses a primer and probe composition and a kit for detecting a chrysanthemum B virus, and a detection method and application of the primer and probe composition, the kit, the detection method and the application of the primer and probe composition, the rapid detection of the chrysanthemum B virus is completed within 5 minutes through the combination of recombinase polymerase amplification (RPA) and lateral flow test strip (LFD) technologies and the combination of isothermal amplification and visual detection advantages, and the sensitivity is 1000 times of that of a conventional RT-PCR method. And the method does not depend on complex instruments and equipment and is suitable for field and basic laboratory operation. The primer and the probe are designed for a conserved domain of a chrysanthemum B virus genome, do not have cross reaction with common chrysanthemum viruses such as chrysanthemum dwarf viroid CSVd, chrysanthemum chlorotic mottle viroid CChMVd, tobacco mosaic virus TMV and the like and host plants, and are high in specificity. A detection result is directly interpreted through color development of the test strip, and the stability is high. The method is highly effective in chrysanthemum seedling detoxification identification, early warning of diseases and large-scale field screening, and provides technical support for accurate prevention and control of chrysanthemum virus diseases and healthy seed industry development.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant virus detection, and in particular relates to a primer and probe combination, a kit, a detection method and an application thereof for detecting chrysanthemum B virus. Background Art

[0002] Chrysanthemum (Chrysanthemum morifolium) is an important economic crop in my country. Viral diseases caused by Chrysanthemum virus B (CVB) are a common type of viral disease in chrysanthemum production, seriously affecting the yield and quality of chrysanthemum.

[0003] At present, the detection technology of CVB has problems such as insufficient sensitivity and complex operation. Although traditional detection technologies such as polymerase chain reaction (PCR) and its derivative technologies (such as RT-PCR) are widely used, they rely on thermal cyclers for temperature control (such as 94°C denaturation, 72°C extension, etc.), require complex equipment and are time-consuming (usually 1.5-2 hours). Although the loop-mediated isothermal amplification (LAMP) technology has gotten rid of the limitations of thermal cyclers and can complete amplification at a constant temperature (40-60°C), its reaction time still takes about 1 hour, and false positives are easily caused by aerosol contamination during the amplification process. The primer design is complex (4-6 specific primers are required), and it is highly dependent on the target sequence, making it difficult to adapt to rapid viral mutations or complex infection scenarios. In addition, enzyme-linked immunosorbent assay (ELISA) relies on the labeling and binding of virus-specific antibodies. Although it has been commercialized, the cost of antibody development is high, and it cannot detect virus-like viruses without capsid proteins. The sensitivity is insufficient (usually the detection limit is 10 -2 ~10 -3 μg / mL), which is easily interfered by impurities in the sample and leads to nonspecific color development, making it difficult to meet the needs of early field diagnosis. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a primer and probe combination and a supporting kit for detecting chrysanthemum B virus that can be used efficiently, sensitively and portablely; another purpose of the present invention is to provide a method for detecting chrysanthemum B virus to solve the problems of traditional technology and equipment dependence, long time consumption and high false positive rate.

[0005] Technical solution: The primer and probe combination for detecting chrysanthemum B virus of the present invention comprises:

[0006] Upstream primer: 5′-GCCAGGAACATAAATCGTCTGACTCAACTC-3′;

[0007] Downstream primer: 5′-[Biotin]-CTTCCACAGGAAATCGGTGGATACTTGACTG-3′;

[0008] Probe:

[0009] 5'-[FAM]-GAATGTGCACGTGACGAATATGGCTCTAGAG[THF]TAGGAAGGCCT GCTC-[C3spacer]-3'.

[0010] Preferably, the 5' end of the downstream primer is labeled with Biotin; the fluorescent group at the 5' end of the probe is FAM, and the fluorescent group at the 3' end of the probe is C3 spacer.

[0011] The chrysanthemum B virus RT-RPA-LFD detection kit of the present invention comprises a primer and probe combination for detecting chrysanthemum B virus.

[0012] Preferably, the apparatus further comprises a plant RNA extraction reagent, an RNA constant temperature rapid amplification reagent, a nucleic acid detection test strip, and a reverse transcription kit.

[0013] The method for detecting chrysanthemum B virus of the present invention is to detect chrysanthemum B virus in a chrysanthemum sample using the primer and probe combination.

[0014] Preferably, the method comprises the following steps:

[0015] (1) Collect chrysanthemum samples and extract RNA;

[0016] (2) using the RNA obtained in step (1) as a template, and performing an RT-RPA amplification reaction using the primer and probe combination for detecting chrysanthemum B virus;

[0017] (3) Dilute the amplified product and add it dropwise to the test strip, and determine the result based on the color development of the quality control line and the test line.

[0018] Preferably, in step (2), the RNA extracted in step (1) is subjected to RT-RPA amplification reaction using a primer and probe combination for detecting chrysanthemum B virus, and the system contains Abuffer, forward primer, reverse primer, probe, B buffer, and RNA template; the RT-RPA amplification reaction procedure is: 42° C., 5 min.

[0019] Further preferably, in step (2), the RNA extracted in step (1) is subjected to RT-RPA amplification reaction using a primer and probe combination for detecting chrysanthemum B virus. The 50 μL system contains 29.4 μL of Abuffer, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 0.3 μL of 10 μM probe, 2.0 μL of B buffer, 1 μL of RNA template, and double-distilled water to 50 μL.

[0020] Preferably, in step (3), the reaction product obtained in step (2) is added to ddH2O, mixed and then the diluted amplification product is dripped into the sample well of the test strip, the control line and the detection line are recorded, and the test result is determined.

[0021] Further preferably, in step (3), 10 μL of the reaction product obtained in step (2) is added to 190 μL of ddH2O, mixed, and 70 μL of the diluted amplification product is dropped into the sample well of the test strip. The control line and the detection line are recorded within 5 minutes to determine the test result.

[0022] Preferably, the method for determining the test results is as follows:

[0023] Positive: A blue stripe appears on the control line of the test strip and a red stripe appears on the test line;

[0024] Negative: A blue band appears on the control line of the test strip, but no band appears on the test line;

[0025] Invalid: No bands appear on either the control line or the test line of the test strip.

[0026] The primer and probe combination for detecting chrysanthemum B virus of the present invention is used in detecting or assisting in detecting chrysanthemum B virus, or in preparing a product for detecting or assisting in detecting chrysanthemum B virus.

[0027] The present invention relates to the use of the chrysanthemum B virus RT-RPA-LFD detection kit in detecting or assisting in the detection of chrysanthemum B virus, or in preparing a product for detecting or assisting in the detection of chrysanthemum B virus.

[0028] Principle of the invention: Recombinase Polymerase Amplification (RPA) technology can achieve exponential amplification of target sequences at room temperature through recombinase-mediated DNA single-strand binding and chain displacement reactions, without the need for complex thermal cycling equipment, and is particularly suitable for rapid field testing. Lateral flow dipstick (LFD) is a visual detection tool that can achieve a "one-step" operation for result interpretation through the specific binding of colloidal gold-labeled probes to the amplified products, combining convenience and accuracy. Combining RPA with LFD (PRA-LFD) can not only circumvent the traditional PCR's dependence on precision instruments, but also reduce the risk of contamination through a closed reaction system, while improving detection throughput and cost-effectiveness.

[0029] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: the present invention integrates recombinase polymerase amplification (RPA) and lateral flow test strip (LFD) technology to achieve isothermal rapid detection, complete detection within 5 minutes, and the sensitivity is 1000 times that of RT-PCR; at the same time, the system has a specific specificity for detecting CVB, and has no cross-reaction with common chrysanthemum viruses (such as chrysanthemum dwarf virus, chrysanthemum chlorotic mottle virus, tomato sterility virus, etc.) and host plants. The present invention overcomes the core problems of insufficient sensitivity, cumbersome operation, and strong equipment dependence of traditional methods. It is suitable for rapid field screening and provides an efficient, economical and reliable solution for the early diagnosis of chrysanthemum virus B (CVB), seed health management, complex infection monitoring, and precise prevention and control. It has broad application prospects and industrial promotion value, and promotes the sustainable development of the chrysanthemum industry in a green and efficient direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the result of CVB capsid protein sequence alignment;

[0031] Figure 2 Screening for reaction time of RT-RPA-LFD assay;

[0032] Figure 3 Screening reaction temperature for RT-RPA-LFD detection;

[0033] Figure 4 The sensitivity of RT-PCR detection of Chrysanthemum virus B (CVB);

[0034] Figure 5 The sensitivity of RT-RPA-LFD detection for Chrysanthemum virus B (CVB);

[0035] Figure 6 Specificity for RT-RPA-LFD detection of Chrysanthemum virus B (CVB). DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] Example 1

[0038] 1. Materials and Methods

[0039] 1.1 Materials

[0040] The experimental materials included chrysanthemum leaves infected with Chrysanthemum virus B (CVB), Chrysanthemum stunt viroid (CSVd), Chrysanthemum chlorotic mottle viroid (CChMVd), and Tobacco Mosaic Virus (TMV), as well as leaves from virus-free chrysanthemum tissue-cultured test tube plantlets. All materials were provided by the inventor's laboratory.

[0041] The rapid universal plant RNA extraction kit was purchased from Beijing Huayueyang Biotechnology Co., Ltd.; the RNA constant temperature rapid amplification kit (nfo enzyme type) was purchased from Changzhou Anpu Future Biotechnology Co., Ltd.; the nucleic acid detection test strips were purchased from Changzhou Anpu Future Biotechnology Co., Ltd.; and the reverse transcription kit (Evo M-MLVPlus cDNA synthesis kit) was purchased from Aikerui Bioengineering Co., Ltd.

[0042] 1.2 Methods

[0043] 1.2.1 Preparation of total RNA samples

[0044] RNA was extracted from the samples using the Beijing Huayueyang Rapid Universal Plant RNA Extraction Kit according to the kit instructions. The extracted RNA was stored at -80°C until use.

[0045] 1.2.2 Design of primers and probes

[0046] The CVB capsid protein sequence of the sample was amplified and TA cloned. The positive clones were then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were compared, and primers and probes were designed in the conserved regions. According to the RT-RPA primer design principles, sequence analysis was performed using software such as SnapGene, SeqHunter2, and MEGA-X. The RPA detection primer design principles are as follows: primer size 25-35 bp, no secondary structure and single-base repeat sequence, and target fragment size 100-500 bp. The corresponding sequences are shown in Table 1 (where 5'FAM is the full name of 5'carboxyfluorescein, C3 spacer is a modification group, and 5'-Biotin refers to the addition of a biotin label at the 5' end). Primers and probes were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0047] Table 1 Primers used in the present invention

[0048]

[0049] 1.2.3 Establishment and Optimization of RT-RPA-LFD Detection System

[0050] Using RNA from CVB-infected (+) samples, virus-free vaccine RNA (-), and sterile ultrapure water (-) as templates, the RT-RPA-nfo (test strip) kit and modified preferred primers and probes were used to optimize the reaction system. Six reaction time gradients (5, 10, 15, 20, 25, and 30 min) and three reaction temperature gradients (37, 42, and 45°C) were set. Based on the test results, the optimal reaction time and temperature were screened to establish the RT-RPA-LFD detection system.

[0051] RT-RPA-nfo (test strip type) amplification reaction system (total volume 50 μL): Add 29.5 μL of Abuffer, 1 μL of modified upstream and downstream primers (10 μmol·L-1), 0.3 μL of probe (10 μmol·L-1), 1 μL of nucleic acid (197 μg / mL), and 2.0 μL of B buffer to the reaction tube in sequence; directly aspirate 10 μL of the liquid after the reaction is completed, add 190 μL of deionized water to dilute and mix, then dispense 70 μL of the diluted amplification product into the sample well of the nucleic acid detection test strip. Record the control line and test line within 5 minutes to determine the test result.

[0052] Method for interpreting nucleic acid test paper results:

[0053] Positive (+): A blue band appears on the control line (C line) of the test strip; a red band appears on the test line (T line);

[0054] Negative (-): A blue band appears on the control line (C line) of the test strip, and there is no band on the test line (T line);

[0055] Invalid: No bands appear on either the control line (C line) or the test line (T line) of the test strip.

[0056] 1.2.4 RT-RPA-LFD sensitivity test

[0057] The total RNA nucleic acid concentration of chrysanthemum samples containing CVB was adjusted to 100 ng / μL, and then the solution was diluted in a 10-fold gradient, and 6 10 -0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The sensitivity of the established method was determined using the total RNA of positive chrysanthemum diluted in a gradient as a template, and water was set as a template as a negative control. RT-PCR and RT-RPA-LFD were used in parallel to compare the sensitivity of different methods.

[0058] 1.2.5 RT-RPA-LFD specificity test

[0059] RNA from samples containing CVB served as a positive control, and RNA from virus-free seedlings served as a negative control. The optimized RT-RPA-LFD method was used for both RT-RPA-LFD and RT-RPA electrophoresis detection, using RNA extracted from samples containing CSVd, TMV, and CChMV as templates. The specificity of the RT-RPA-LFD assay was determined based on the results.

[0060] 2. Results and Analysis

[0061] 2.1 Establishment and optimization of the RT-RPA-LFD detection system

[0062] 2.1.1 Design of primers and probes

[0063] The sequencing results showed that the amplified gene size was 945 bp. Subsequently, the measured sequences were compared and analyzed, and the results were as follows ( Figure 1 ), conserved regions were selected to design RT-RPA-LFD primers and probes.

[0064] 2.1.1 Reaction system optimization

[0065] The test results showed that the results can be obtained after 5 minutes of RT-RPA amplification. As the amplification time increases, the red color of the strip in the test area gradually deepens. However, when the gene amplification lasts for more than 10 minutes, the negative control will produce a false positive ( Figure 2 ), so the ideal time for gene amplification during detection is 3 to 5 minutes.

[0066] The temperature screening results showed that when the amplification was performed at several gradient temperatures from 37°C to 45°C for 5 minutes, false positives were found at both 37°C and 45°C ( Figure 3 Therefore, the recommended temperature for the kit is 42°C.

[0067] 2.2 Evaluation of the RT-RPA-LFD detection system

[0068] 2.2.1 Sensitivity test

[0069] After the concentration of total RNA nucleic acid and cDNA nucleic acid of CVB sample was adjusted to 100ng / μL, it was diluted in 10-fold gradient. The original solution and the dilution were tested by different methods. The test results showed that when the original solution was diluted to 10 -1 When the stock solution was diluted to 10 -4 When RT-PCR did not detect CVB, the RT-RPA-LFD detection area still showed a red band, indicating that CVB was detected ( Figure 4-5 The results showed that the detection sensitivity of RT-RPA-LFD was 1000 times that of RT-PCR.

[0070] 2.2.2 Specificity detection

[0071] The test results showed that RT-RPA-LFD detection showed that samples infected with CVB were positive, while the rest of the samples were negative. This shows that the established RT-RPA-LFD detection method is specific for CVB detection and has no cross-reaction with Chrysanthemum stunt viroid (CSVd), Chrysanthemum chlorotic mottle viroid (CChMVd) and Tobacco Mosaic Virus (TMV), showing strong specificity for CVB. Figure 6 ).

Claims

1. A primer and probe combination for detecting chrysanthemum B virus, characterized in that: Include: Upstream primer: 5′-GCCAGGAACATAAATCGTCTGACTCAACTC-3′; Downstream primer: 5′-[Biotin]-CTTCCACAGGAAATCGGTGGATACTTGACTG-3′; Probe: 5'-[FAM]-GAATGTGCACGTGACGAATATGGCTCTAGAG[THF]TAGGAAGGCCT GCTC-[C3spacer]-3'.

2. A chrysanthemum B virus RT-RPA-LFD detection kit, characterized in that: A primer and probe composition for detecting chrysanthemum B virus comprising the primer and probe composition of claim 1.

3. The RT-RPA-LFD detection kit for Chrysanthemum B virus according to claim 2, characterized in that: It also includes plant RNA extraction reagents, RNA constant temperature rapid amplification reagents, nucleic acid detection test strips, and reverse transcription kits.

4. A method for detecting chrysanthemum B virus, characterized in that: The primer and probe combination according to claim 1 is used to detect chrysanthemum B virus in chrysanthemum samples.

5. The method for detecting chrysanthemum virus B according to claim 4, characterized in that: The following steps are involved: (1) Collect chrysanthemum samples and extract RNA; (2) using the RNA obtained in step (1) as a template, and performing an RT-RPA amplification reaction using the primer and probe composition for detecting chrysanthemum B virus according to claim 1; (3) Dilute the amplified product and add it dropwise to the test strip, and determine the result based on the color development of the quality control line and the test line.

6. The method for detecting chrysanthemum virus B according to claim 5, characterized in that: In step (2), the RNA extracted in step (1) is subjected to RT-RPA amplification reaction using a primer and probe combination for detecting chrysanthemum B virus. The system contains Abuffer, forward primer, reverse primer, probe, B buffer, and RNA template. The RT-RPA amplification reaction procedure is: 42° C., 5 min.

7. The method for detecting chrysanthemum virus B according to claim 5, characterized in that: In step (3), the reaction product obtained in step (2) is added to ddH2O, mixed and then the diluted amplification product is dripped into the sample well of the test strip, the control line and the test line are recorded, and the test result is determined.

8. The method for detecting chrysanthemum virus B according to claim 5, characterized in that: The method for determining the test results is as follows: Positive: A blue stripe appears on the control line of the test strip and a red stripe appears on the test line; Negative: A blue band appears on the control line of the test strip, but no band appears on the test line; Invalid: No bands appear on either the control line or the test line of the test strip.

9. Use of the primer and probe combination for detecting chrysanthemum B virus according to claim 1 in detecting or assisting in the detection of chrysanthemum B virus, or in preparing a product for detecting or assisting in the detection of chrysanthemum B virus.

10. Use of the RT-RPA-LFD detection kit for Chrysanthemum B virus according to claim 2 or 3 in detecting or assisting in the detection of Chrysanthemum B virus, or in preparing a product for detecting or assisting in the detection of Chrysanthemum B virus.

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