Alfalfa verticillium detection method based on enzyme-mediated double index amplification technology and detection kit thereof

Abstract

The invention discloses a method for detecting verticillium medicaginis based on an enzyme-mediated double-index amplification technology and a detection kit of the verticillium medicaginis. The invention belongs to the field of biological detection, and particularly relates to a method for detecting verticillium medicaginis based on an enzyme-mediated double-index amplification technology and a detection kit of the verticillium medicaginis. The composition for detecting medicago sativa verticillium comprises a primer F1, a primer R1 and an RNA probe, the nucleotide sequence of the primer F1 is as shown in SEQ ID No: 1, the nucleotide sequence of the primer R1 is as shown in SEQ ID No: 7, and the nucleotide sequence of the RNA probe is as shown in SEQ ID No: 15. The rapid visual detection technology for the verticillium medicaginis EnDEA, which is researched and developed by the invention, is high in specificity, rapid and sensitive, and convenient for detection personnel to carry out rapid detection on the spot, so that the detection time is greatly shortened, the detection cost is reduced, and an important reference is provided for rapid detection of the verticillium medicaginis.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and particularly relates to a detection method of Verticillium acetalicum based on enzyme-mediated double exponential amplification technology and a detection kit thereof. Background Art

[0002] Alfalfa is one of the important economic sources for herders and pastures. It contains a large amount of protein, dietary fiber, various minerals and other nutrients, and is one of the high-quality forage grasses. In addition to being used to feed livestock, alfalfa can also be used as a soil and water conservation and slope protection plant. In gardens, it is often used as a greening grass seed for saline-alkali land and barren land and as a landscape wildflower grass seed. In recent years, alfalfa wilt has occurred widely in various pastoral areas, and has spread rapidly, causing a significant reduction in production or even a complete crop failure, bringing serious economic losses to growers. The pathogen that causes this disease is Verticillium lucerne, which is sporadically distributed in diseased plants and its early symptoms are easily confused with other causes such as yellowing caused by poor environmental conditions, which increases the difficulty of identification. There is an urgent need to establish accurate, rapid and efficient detection technology that is easy to operate in the field and shortens the time for technicians to determine the cause of the disease and take scientific symptomatic prevention and control measures.

[0003] Traditional diagnostic methods for alfalfa Verticillium wilt are primarily based on disease symptoms and morphological identification of the pathogen. This requires the isolation, purification, and culture of diseased tissue, as well as certain professional skills for identification. However, the disease has hidden symptoms, and the typical chlorotic "V" symptom of leaves rarely appears in the field, which can easily lead to misdiagnosis in the field. Currently, the current entry-exit inspection and quarantine industry standard "SN / T 1145-2014" Quarantine and Identification Method Standard for Verticillium wilt of Alfalfa - Real-time Fluorescence PCR stipulates that when the positive control, negative control, and blank control results are normal, the qualitative PCR specific amplification product fragment size of the alfalfa Verticillium wilt pathogen is 330bp, and the real-time fluorescence PCR Ct value is less than or equal to 36. Large-scale fluorescence PCR instruments are inconvenient for testing in the field and at ports, and the detection time is relatively long. Tian Qing et al. used β-tubulin (TUB) as a target gene and applied LAMP detection technology to a system for detecting Verticillium nigra. They successfully detected the pathogen in diseased alfalfa plant tissue and in alfalfa spores added to simulated soil. However, LAMP primer design is complex and aerosol contamination can easily lead to false positives. Qian Yike et al. developed a multiplex PCR based on the TUB gene for dual detection of Verticillium nigra and Verticillium dahliae, but base pairing interference or significant differences in annealing temperatures between primers can occur. Summary of the Invention

[0004] The main problem to be solved by the present invention is how to quickly detect Verticillium lucerne.

[0005] In order to solve the above problems, the present invention provides a composition for detecting Verticillium acetalicum.

[0006] The composition for detecting Verticillium acetalicum provided by the present invention comprises a primer F1, a primer R1 and an RNA probe, the nucleotide sequence of the primer F1 is shown in SEQ ID No: 1, the nucleotide sequence of the primer R1 is shown in SEQ ID No: 7, and the nucleotide sequence of the RNA probe is shown in SEQ ID No: 15.

[0007] In the above composition, the 5' end of the RNA probe is labeled with a reporter group, and the 3' end is labeled with a quencher group.

[0008] Furthermore, the fluorescent group can be selected from at least one of FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705 or Texas Red.

[0009] Furthermore, the quenching group can be selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB, and Dabcy1.

[0010] In the above composition, the reporter group is FAM, and the quencher group is BHQ1.

[0011] The present invention also provides a kit for detecting Verticillium acetalicum, wherein the kit comprises the composition described above.

[0012] Furthermore, the kit also includes detection reagents required for enzyme-mediated dual exponential amplification nucleic acid detection technology.

[0013] Furthermore, the kit also contains a basic dry powder and an activator. The basic dry powder includes the lyophilized product of the enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase, and signal amplifier enzyme, and a lyophilization protectant. The activator can be NTP (nucleoside triphosphate) and a buffer.

[0014] The kit may further include a nucleic acid extraction reagent, which is used to extract nucleic acids from a biological sample (sample to be tested).

[0015] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0016] The present invention also provides use of the above-mentioned composition in detecting Verticillium lucerne.

[0017] The present invention also provides use of the above-mentioned composition in preparing a product for detecting Verticillium acetalicum.

[0018] The product described herein can be a reagent, a kit, a chip, a test paper or a test card.

[0019] The use of the above-mentioned kit in detecting Verticillium lucerne also falls within the scope of protection claimed by the present invention.

[0020] The present invention also provides a method for detecting Verticillium medicagogue, which comprises using the composition or the kit described above to perform an amplification reaction on a sample to be tested, and determining whether the sample to be tested contains Verticillium medicagogue or is Verticillium medicagogue based on the amplification result, wherein the amplification reaction is performed based on an enzyme-mediated double exponential amplification nucleic acid detection technology.

[0021] The above method comprises the following steps:

[0022] A1) extracting DNA from the sample to be tested;

[0023] A2) using the test sample DNA as a template, performing enzyme-mediated dual exponential amplification nucleic acid detection using the composition described in the preceding claims to form an amplification curve;

[0024] A3) Determine the test result based on the Ct value.

[0025] The result determination method is as follows:

[0026] 1) If the Ct value is less than or equal to 30, the sample to be tested contains Verticillium medicagogue or is Verticillium medicagogue;

[0027] 2) If the Ct value is greater than 30, the sample to be tested does not contain Verticillium medicagogue or is not Verticillium medicagogue.

[0028] Furthermore, the smaller the Ct value and the larger the peak value, the better the detection effect or the higher the DNA concentration.

[0029] In a specific embodiment, the reaction system for the enzyme-mediated dual exponential amplification of nucleic acid is: 1 μL (10 μmol / L) of upstream primer F1, 1 μL (10 μmol / L) of downstream primer R1, 1 μL (1 μmol / L) of RNA probe N3, 7 μL of DNA of the sample to be tested, 10 μL of activation solution and basic dry powder.

[0030] The basic dry powder comprises enzymes required for detection: freeze-dried products of DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase and signal amplification enzyme, and freeze-dried protective agent.

[0031] The activator may be NTP (nucleoside triphosphate) and a buffer.

[0032] The reaction conditions of the enzyme-mediated dual exponential amplification of nucleic acid are: constant temperature at 42° C., collecting a signal once per minute, and collecting a total of 30 signals.

[0033] In this study, multiple candidate genes for Verticillium alfalfa were amplified and sequenced, ultimately selecting the SACR region as a target. By comparing the SACR sequence with the gene sequences of other Verticillium species, upstream and downstream primers and RNA primers for the enzyme-mediated duplex exponential amplification (EmDEA) assay were designed. Screening of these primers identified the optimal combination, N3F1R1, and a rapid visual detection technique for V. alfalfa using EmDEA was established. Experiments with V. alfalfa and its related species demonstrated that this method exhibited high specificity, detecting only V. alfalfa. Furthermore, this method is rapid and sensitive, facilitating rapid testing in the field. The results allow for rapid sample processing, significantly reducing testing time and costs, and providing an important reference for the rapid detection of V. alfalfa. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 These are the screening results of RNA probes for detecting Verticillium acetalicum.

[0035] Figure 2 These are the screening results of downstream primers for detecting Verticillium lucerne.

[0036] Figure 3 These are the screening results of upstream primers for detecting Verticillium lucerne.

[0037] Figure 4 This is a study on the specificity of the EmDAE detection technology established in the present invention.

[0038] Figure 5 Sensitivity study of the EmDAE detection technology established in the present invention.

[0039] Figure 6 To simulate the detection results of Verticillium lucerne samples. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0041] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0042] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0043] The following examples are described in: Li Mingle, Lei Rong, Yang Li, et al. Establishment of a Rapid Detection Method for Verticillium Medicago AV02 using RPA / CRISPR-Cas12a [J]. Acta Mycologica Sinica, 2024, 43(09): 83-98. DOI: 10.13346 / j.mycosystema.230203.). The public may obtain the biological material from the applicant for use only in repeating the experiments of the present invention and may not be used for other purposes.

[0044] The strains in Table 2 of the following examples have been recorded in: [1] Guo Lixin, Zhang Xianglin, Chen Xianfeng, et al. Detection method of black and white Verticillium based on TaqMan MGB probe [J]. Plant Quarantine, 2017, 31(01): 32-36.); [2] Duan Weijun, Li Xuelian, Lv Yan, et al. Rapid detection method of long-spored Verticillium by TaqMan-MGB probe real-time fluorescence PCR [J]. Plant Protection, 2023, 49(04): 224-232+255. The public may obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0045] The primers and probes used in the following examples were designed based on the SACR sequence of Verticillium truncatum determined in our laboratory and published in GenBank (Genbank No. DQ333342.1, updated on September 15, 2010). The primer and probe sequence information is shown in Table 1. DNA primers were synthesized by BGI, and RNA probes were synthesized by Suzhou Jingrui Biotechnology Co., Ltd. The RNA probes were labeled with the reporter group FAM at the 5' end and the quencher group BHQ1 at the 3' end.

[0046] Table 1. Primer and probe sequence information

[0047]

[0048]

[0049] Example 1: Establishment of EmDEA Detection Method for Verticillium lucerne

[0050] 1. DNA extraction

[0051] V. alfalfa AV02 was cultured on PDA medium at 25°C for 10 days. Mycelium from the culture plate was scraped and DNA was extracted using a nucleic acid extraction kit. The extracted DNA was analyzed for concentration using NanoDrop and stored at -80°C for future use.

[0052] 2. RNA probe screening

[0053] The enzyme-mediated dual exponential amplification (EmDEA) reaction system (20 μL) used in this study consisted of 1 μL of DNA upstream primer (10 μmol / L), 1 μL of DNA downstream primer (10 μmol / L), 1 μL of RNA probe (1 μmol / L), 7 μL of DNA, 10 μL of activation solution, and one tube of dry powder. The dry powder and activation solution were from the Basic Fluorescence Constant Temperature Amplification Detection Kit (RR032) from Suzhou Jingrui Co., Ltd. The reaction temperature was constant at 42°C, and the signal was collected once every minute for a total of 30 times.

[0054] Using AV02 as a DNA template, RNA probe screening was performed. First, the middle primers F2, R2, F2, and R3 were cross-linked to obtain four primer combinations: F2R2, F2R3, F3R2, and F3R3. These four primer combinations were cross-linked with the six RNA probes N1, N2, N3, N4, N5, and N6 to form a total of 24 primer-probe combinations.

[0055] Each of the 24 combinations was tested on the instrument. After the amplification test results were available, the six probe pairs from the same primer set were compared, and the RNA probe with the earliest peak time and the highest endpoint fluorescence value was selected. The four primer combinations with the lowest Ct value and the highest endpoint fluorescence value were then screened for subsequent screening experiments based on the selected probes. Three replicates and one negative control were set up for each primer-probe combination. The negative control used nuclease-free water as the template, while other conditions remained unchanged. The selected combination required the negative control to have no fluorescent signal.

[0056] The results are as follows: All RNA probes in the four primer combinations produced amplification signals, but RNA probe N3 had an earlier peak and a smaller Ct value than the other RNA probes, so N3 was selected as the target RNA probe. Figure 1 Among them, the Ct value of the F3R2 group was smaller, the fluorescence value was the highest, and the corresponding negative control had no signal, so N3F3R2 was selected for subsequent experiments.

[0057] 3. Initial round of DNA downstream primer screening

[0058] Downstream primer screening was performed using AV02 as the DNA template, N3 as the RNA probe, and F3 as the DNA upstream primer.

[0059] Six DNA downstream primers (R1, R2, R3, R4, R5, and R6) were tested in combination with the immobilized RNA probe N3 and the DNA upstream primer F3. The DNA downstream primer with the lowest Ct value and the highest endpoint fluorescence value was selected. Three replicates and a negative control were set for each primer-probe combination. The negative control used nuclease-free water as the template. All other conditions remained unchanged. The selected combination required the negative control to show no fluorescence signal.

[0060] Depend on Figure 2 It can be seen that the Ct value of downstream primer R1 is smaller, which is 8.64, and the fluorescence value is the highest. In addition, the corresponding negative control has no signal. Therefore, primer R1 is selected as the downstream primer.

[0061] 4. Initial round of DNA upstream primer screening

[0062] Upstream primer screening was performed using AV02 as the DNA template, N3 as the RNA probe, and R1 as the DNA downstream primer.

[0063] Six DNA upstream primers (F1, F2, F3, F4, F5, and F6) were tested in combination with an immobilized RNA probe and a DNA downstream primer. The DNA downstream primer with the lowest Ct value and the highest endpoint fluorescence value was selected. Three replicates and a negative control were set for each primer-probe combination. The negative control used water as the template, and all other conditions remained unchanged. The selected combination required the negative control to show no fluorescence signal.

[0064] Depend on Figure 3 It can be seen that the Ct value of upstream primer F1 is smaller, which is 9.26, and the fluorescence value is the highest. In addition, the corresponding negative control has no signal. Therefore, primer F1 is the upstream primer screened in this experiment.

[0065] Based on the above three screening experiments, N3F1R1 was finally selected as the primer-probe combination for this experiment.

[0066] The optimal isothermal fluorescence detection reaction system (20 μL) established in this example is as follows: upstream primer F1 1 μL (10 μmol / L), downstream primer R1 1 μL (10 μmol / L), RNA probe N3 1 μL (1 μmol / L), sample DNA to be tested 7 μL, activation solution 10 μL and a tube of basic dry powder.

[0067] The basic dry powder includes the enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase and signal amplifier enzyme freeze-dried products and freeze-dried protective agent.

[0068] The activator can be NTP (nucleoside triphosphate) and a buffer.

[0069] The activation solution and basic dry powder were derived from the basic fluorescence constant temperature amplification detection kit (RR032) of Suzhou Jingrui Co., Ltd.

[0070] The optimal isothermal fluorescence detection reaction conditions are: 42℃, reaction time: 30min.

[0071] The result is determined as follows: if the Ct value is less than or equal to 30, it is determined that the sample to be tested contains Verticillium medicagogue; if the Ct value is greater than 30, it is determined that the sample to be tested does not contain Verticillium medicagogue.

[0072] Example 2: Study on the specificity and sensitivity of the EmDAE detection technology established by the present invention

[0073] 1. Specificity experiment

[0074] The specificity experiment was performed using the N3F1R1 combination as the primer-probe combination required for the experiment and the DNA of the strains shown in Table 1 as the template.

[0075] The strains used in the specificity experiment were 4 strains of Verticillium medicagogue and 32 other strains (Table 2). The reaction system was the same as the optimal reaction system in Example 1. The detection method was the same as Example 1.

[0076] Table 2. Test strain information

[0077]

[0078]

[0079] The results are as follows Figure 4 As shown in the results, all four strains of Verticillium medicagogue were effectively detected, while the negative control group strains and their blanks could not be detected, indicating that the N3F1R1 primer-probe combination had good specificity.

[0080] 2. Sensitivity test

[0081] A sensitivity experiment was conducted using AV02 as the DNA template and N3F1R1 as the primer-probe combination. The AV02 DNA concentration was determined to be 100 ng / μL. The DNA was diluted 10-fold with water across six concentration gradients, with three replicates per gradient.

[0082] The results showed that the lowest dilution gradient that the primer probe could detect was 10 -4 , that is, the sensitivity of this combination is 10pg of total DNA content in 20μL reaction system ( Figure 5 ).

[0083] Example 3: Application of EmDAE Detection Method in Detecting Verticillium Medicago Disease in Samples

[0084] Purchase alfalfa hay (Bickbear JD flagship store, item number B20230344), select 2 g of healthy alfalfa stems, disinfect with 75% alcohol, cut the healthy stems into small pieces, and mix them with 100 mg of mycelium of Verticillium medicagogue grown for 5-7 days to form sample 1.

[0085] Healthy stems were cut into small pieces and mixed with 200 mg of mycelium of Verticillium medicagogue AV02 grown for 5-7 days to form sample 2;

[0086] Healthy stems were cut into small pieces and mixed with 300 mg of mycelium of Verticillium medicagogue AV02 grown for 5-7 days to form sample 3;

[0087] Nuclease-free water was used as the NTC group.

[0088] AV02 was used as the positive template.

[0089] The mixed sample was placed in a 50 mL grinding jar, and one sterile steel ball was added. The sample was then frozen overnight at -80°C. The sample was then thoroughly ground in a high-throughput tissue disruptor (Retsch, MM400). DNA was extracted using an automated nucleic acid extraction instrument according to the instructions for the MAG Plant DNA Extraction Kit. The extracted DNA was then tested for Verticillium truncatula using the optimized EmDAE method described in Example 1.

[0090] The test results showed that: with AV02 as the positive control, the test results of the three samples of Verticillium Medicago truncatula DNA were all positive, with small Ct values ​​and high peak values ​​( Figure 6 ). This shows that the N3F1R1 combination has a good detection effect and can be used in actual sample detection.

[0091] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A composition for detecting Verticillium acetalicum, characterized in that: The composition includes primer F1, primer R1 and an RNA probe, the nucleotide sequence of the primer F1 is shown as SEQ ID No: 1, the nucleotide sequence of the primer R1 is shown as SEQ ID No: 7, and the nucleotide sequence of the RNA probe is shown as SEQ ID No:

15.

2. The composition according to claim 1, characterized in that The 5' end of the RNA probe is labeled with a reporter group, and the 3' end is labeled with a quencher group.

3. The composition according to claim 2, characterized in that The reporter group is FAM, and the quencher group is BHQ1.

4. A kit for detecting Verticillium lucerne, characterized in that: The kit comprises the composition according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also contains basic dry powder and an activator; the basic dry powder includes enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase and freeze-dried products of signal amplifier enzyme and a freeze-dried protective agent; the activator can be nucleoside triphosphate and buffer.

6. Use of the composition according to any one of claims 1 to 3 in detecting Verticillium acetalicum.

7. Use of the composition according to any one of claims 1 to 3 in the preparation of a product for detecting Verticillium acetalicum.

8. Use of the kit according to claim 4 or 5 in detecting Verticillium acetalicum.

9. A method for detecting Verticillium lucerne, characterized in that: The method comprises performing an amplification reaction on a sample to be tested using the composition according to any one of claims 1 to 3 or the kit according to claim 4 or 5, and determining whether the sample to be tested contains Verticillium medicagogue or is Verticillium medicagogue according to the amplification result, wherein the amplification reaction is performed based on an enzyme-mediated dual exponential amplification nucleic acid detection technology.

10. The method according to claim 9, characterized in that The method comprises the following steps: A1) extracting DNA from the sample to be tested; A2) using the test sample DNA as a template, performing enzyme-mediated dual exponential amplification nucleic acid detection using the composition of any one of claims 1 to 3 to form an amplification curve; A3) Determine the test result based on the Ct value.

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