CN117867152A - Nucleic acid primer group for detecting bacterial wilt, product containing same and application - Google Patents
Nucleic acid primer group for detecting bacterial wilt, product containing same and application Download PDFInfo
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Abstract
The invention discloses a nucleic acid primer group for detecting bacterial wilt, a product containing the same and application of the nucleic acid primer group. The nucleic acid primer group comprises any one of a primer group I for amplifying a bacterial wilt III type secretion system regulatory gene hrpB and a primer group II for amplifying a bacterial wilt flagellin gene flic; the primer group I comprises F3 with nucleotide sequences shown as SEQ ID NO. 1-6 respectively i Primer, B3 i Primers, FIP i Primer, BIP i Primer, LF i Primers and LB i A primer; the primer group II comprises F3 with nucleotide sequences shown as SEQ ID NO 7-12 respectively j Primer, B3 j Primers, FIP j Primers, BIP j Primer, LF j Primers and LB j And (5) a primer. The nucleic acid primer group provided by the invention is used for detecting the bacterial wilt, has the advantages of strong specificity, high sensitivity and visual detection result, and can realize the rapid and accurate detection of the potato bacterial wilt.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a nucleic acid primer group for detecting bacterial wilt, a product containing the same and application of the nucleic acid primer group.
Background
Bacterial wilt is a destructive soil-borne disease caused by bacterial wilt (ralstoniaanaceae). The plant disease is one of the plant diseases with the greatest harm, the greatest distribution and the most serious loss in the world, and no effective chemical pesticide and other control methods exist until now. Bacterial wilt is therefore referred to as "cancer" of the plant. Ralstonia solanacearum (r.dolaaraceum) can infect more than 40 plants of family 200, next to agrobacterium tumefaciens. Is an important limiting factor for the production of many plants such as tomatoes, potatoes, peanuts, sweet potatoes, tobacco, peppers, eggplants, ginger, strawberries, bananas and some precious medicinal materials and flower plants, and is distributed all over the world.
Bacterial wilt of potato is a destructive bacterial disease caused by ralstonia solanacearum (Ralstonia solanacearum), the second largest disease of potato. It has a wide host range, with distribution in the tropical, subtropical and temperate zones. Can be transmitted through soil, irrigation water, plants, seed potatoes and the like. At present, seed potato disease is a main way for long-distance transmission of potato bacterial wilt. In the early stage of disease, the tips and leaves of potato are easy to drop. In the middle and late stages of the disease, the vascular bundles of the plants and potato pieces turn brown. At the end of disease, the diseased plants die, and opalescent bacterial pus appears when the diseased potato is cut to extrude the brown vascular bundles, so that the quality and the value of the potatoes are seriously affected.
The existing detection methods for potato bacterial wilt mainly comprise a visual detection method, a selective separation method, a serological detection method, a molecular biological detection method and the like. The visual inspection method is used for observing the symptoms of potato bacterial wilt, but the traditional method has a plurality of defects, and the latent infected potato seeds cannot be seen. The selection separation method is that KELMAN et al separates the bacterial wilt by using TZC culture medium for the first time in 1954, and the method is simple to operate, but is time-consuming, labor-consuming and inconvenient to produce and apply. Serological detection is a detection method based on the specific binding of antigen and antibody. The enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescent staining (IFAS) are commonly used, and the method is simple, convenient and easy to generate cross reaction, and the reliability of the detection result is influenced by the change of the sample quality and the treatment method. With the rapid development of molecular biology technology and derivative technology thereof in the 80 th century, the accuracy, stability and sensitivity of detection are remarkably improved, and most commonly used are restriction fragment length polymorphism, polymerase Chain Reaction (PCR) and the like. OPINA et al designed the bacterial wilt specific primer Au759f/760r in 1997, and the primer is still used at present due to high accuracy and good specificity. However, this PCR method requires a long amplification time, requires expensive equipment, and cannot be identified on site.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention provides a nucleic acid primer group for detecting bacterial wilt, a product containing the same and application of the nucleic acid primer group. The invention designs the specific LAMP primer based on two different gene sequences of the bacterial wilt and visualizes the primer, and the primer is applied to potato detection, can realize the rapid and accurate detection of the bacterial wilt of the potato, is simple and convenient to operate, can meet the field detection, provides an executable and reasonable-cost bacterial wilt detection method for basic technical departments and enterprises, and enriches the detection means of the bacterial wilt.
In a first aspect of the present invention, there is provided a nucleic acid primer set for detecting bacterial wilt comprising any one of a primer set I for amplifying a bacterial wilt type III secretion system regulating gene hrpB and a primer set II for amplifying a bacterial wilt flagellin gene flic; the primer group I comprises F3 with nucleotide sequences shown as SEQ ID NO. 1-6 respectively i Primer, B3 i Primers, FIP i Primer, BIP i Primer, LF i Primers and LB i A primer; the primer group II comprises F3 with nucleotide sequences shown as SEQ ID NO 7-12 respectively j Primer, B3 j Primers, FIP j Primer, BIP j Primer, LF j Primers and LB j And (5) a primer.
In a second aspect of the present invention, there is provided a loop-mediated isothermal amplification reagent comprising the aforementioned nucleic acid primer set.
In a third aspect of the invention, there is provided a kit for detecting ralstonia solanacearum, the kit comprising the loop-mediated isothermal amplification reagent as described above.
In a fourth aspect of the invention, there is provided the use of a nucleic acid primer set or a reagent or kit as described above for detecting ralstonia solanacearum.
In a fifth aspect of the invention, there is provided the use of a nucleic acid primer set or a reagent or kit as hereinbefore described for the detection of ralstonia solanacearum.
In a sixth aspect of the present invention, there is provided a method for detecting ralstonia solanacearum, comprising: extracting DNA of a sample to be tested can be performed by a simple method, such as using a commercial extraction kit, according to the steps in the specification; carrying out loop-mediated isothermal amplification on DNA of a sample to be detected by adopting the nucleic acid primer group to obtain an amplification product; detection of amplification products can be performed in a variety of ways, depending on the laboratory equipment and the purpose of the experiment, the most common being the use of gel electrophoresis. Alternatively, fluorescent dyes or other special detection reagents may be used to directly observe the amplification results; and judging whether the sample to be detected contains the bacterial wilt or not based on the detection result.
According to a specific embodiment of the present invention, a reaction system for loop-mediated isothermal amplification comprises: 2.5 Mu L10 XThermoPol buffer, 1.5 mu L MgSO 4 、3.5 μL dNTPs、FIP i Primers or FIP j Primer 1 mu L, BIP i Primers or BIP j Primer 1 mu L, F3 i Primers or F3 j Primer 0.5 mu L, B3 i Primer or B3 j Primer 0.5 mu L, LF i Primers or LF j Primer 1 mu L, LB i Primers or LB j 1. Mu.L of primer, 1. Mu. L Bst DNA Polymerase solution, 1. Mu.L of template DNA, make up ddH 2 O to 25. Mu.L.
According to a specific embodiment of the present invention, the loop-mediated isothermal amplification procedure comprises: and (3) performing amplification reaction at the constant temperature, wherein the reaction temperature is 60-65 ℃ and the reaction time is 50-70 min. In this isothermal environment, the primer will bind to the target DNA fragment and amplify by the action of the enzyme.
According to the specific embodiment of the invention, the detection method is to carry out agarose gel electrophoresis on the amplified product, and the judgment standard is as follows: if no target amplification strip appears in the electrophoresis result, judging that the sample to be detected does not contain the bacterial wilt; if the target amplification band appears in the electrophoresis result, the sample to be detected is judged to contain the bacterial wilt.
According to a specific embodiment of the invention, the detection method is to make the amplified product react by contacting with a color developing agent 1000×SYBR Green I, and the judgment standard is: if the reaction liquid is orange, judging that the sample to be detected does not contain the bacterial wilt; if the reaction liquid is green, the sample to be detected is judged to contain the bacterial wilt.
The beneficial effects of the invention are as follows:
the nucleic acid primer group provided by the invention is used for detecting the bacterial wilt, has the advantages of strong specificity, high sensitivity and visual detection result, can realize the rapid and accurate detection of the potato bacterial wilt, fills the gap that the prior art lacks the LAMP primer for detecting the potato bacterial wilt, and can be used for developing products for detecting the potato bacterial wilt in relevant fields.
Drawings
FIG. 1 shows the results of a primer set specificity experiment of the present invention; wherein M is DL 2000 Maker, lane CK is negative control, lanes 1-7 are bacterial strain collection, and lanes 8-14 are early blight respectivelyAlternaria alternataEarly blight and epidemic diseaseAlternaria solaniEpidemic disease in late stagePhytophthora infestansColi, E.coliEnterobacter spBlack moleRhizoctonia solaniDry rot disease of the human bodyFusarium oxysporumAnthracnose of a patientColletotrichum coccodesLane 15 is positive control UW551;
FIG. 2 shows the results of a sensitivity test of the primer set of the present invention; wherein M is DL 2000 Maker, lane CK is negative control, lanes 1-7 are 100×10 respectively 0 ng/μL、100×10 -1 ng/μL、100×10 -2 ng/μL、100×10 -3 ng/μL、100×10 -4 ng/μL、100×10 -5 ng/μL、100×10 -6 ng/μL；
FIG. 3 shows the results of the application effect experiment of the primer set of the present invention; wherein M is DL 2000 Maker, lane CK is a negative control, lanes 1, 4, 5, 6, 7, 9, 10 are potatoes without bacterial wilt, lanes 2, 3, 8 are potatoes with bacterial wilt, and lane 11 is a bacterial wilt positive control UW551.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 DNA extraction of test sample
1. Sample preparation: vascular bundle tissue 100 mg was taken from potato tubers suspected of being infected with bacterial wilt, and taken on site.
2. DNA extraction: the total DNA in the sample was extracted using a commercial DNA extraction kit according to the procedure of the instructions, ensuring that high quality and pure DNA was obtained.
The seed potato sample DNA extraction operation steps are as follows:
2.1 Plant tissue was thoroughly ground by adding liquid nitrogen and fresh plant tissue was weighed to be about 100 mg or dry weight tissue was weighed to be about 30 mg.
2.2 Adding 400 mu L of buffer solution GPS and 10 mu L of RNase A (10 mg/mL) into the ground powder rapidly, vortex and mix uniformly rapidly, placing the centrifuge tube in a water bath at 65 ℃ for 15 min, and inverting the centrifuge tube in the water bath process to mix the sample for several times. Note that if the solution after the cleavage is viscous, the amount of GPS used can be increased appropriately, and at the same time, the amount of buffer GPA used in step 3 is increased, and the final volume ratio of GPS to GPA is 4:1.
2.3 100 μl buffer GPA was added, vortexed and centrifuged for 5 min at 1min at 12,000rpm (13,400X g), the supernatant was transferred to a filter column CS (filter column CS was placed in a collection tube), and then centrifuged for 1min at 12,000rpm (13,400X g), transferring the filtrate to a new centrifuge tube. Note that: if the solution is sticky after the cracking, adding buffer GPA, mixing uniformly by vortex, placing the centrifuge tube on ice for standing for 5 min, and then centrifuging.
2.4 Adding the absolute ethyl alcohol with the same volume, and fully and uniformly mixing, wherein flocculent precipitation can occur.
2.5 The solution obtained in the previous step and the flocculent precipitate were transferred to an RNase-Free adsorption column CR2 (the adsorption column CR2 was placed in a collection tube), centrifuged at 12,000rpm (13,400 Xg) for 1min, the waste liquid was poured off, and the RNase-Free adsorption column CR2 was placed in the collection tube.
2.6 550 mu L deproteinized liquid RD (absolute ethanol is added before use, the volume is added with reference to the label on the bottle) is added to the RNase-Free adsorption column CR2, the mixture is centrifuged at 12,000rpm (13,400 Xg) for 1min, the waste liquid is poured out, and the RNase-Free adsorption column CR2 is placed in a collecting tube.
2.7 700 mu L of rinse solution PW (absolute ethyl alcohol is added before use, the volume is added with reference to the label on a bottle) is added into an RNase-Free adsorption column CR2, the mixture is centrifuged at 12,000rpm (13,400 Xg) for 1min, waste liquid is poured out, and the RNase-Free adsorption column CR2 is placed into a collecting pipe.
2.8 Step 2.7 is repeated.
2.9 The RNase-Free adsorption column CR2 was returned to the collection tube, centrifuged at 12,000rpm (13,400 Xg) for 2 min, the collection tube was discarded, and then the RNase-Free adsorption column CR2 was transferred to a new centrifuge tube and dried at room temperature for 5-10 min. Note that: the residual ethanol inhibits the subsequent enzyme reaction, so that the ethanol is ensured to volatilize cleanly during the air drying process. But also not dried too long to prevent elution of the DNA.
2.10 50-100 mu L of elution buffer TB is added into an RNase-Free adsorption column CR2, the mixture is placed at room temperature for 3-5 min, centrifuged at 12,000rpm (13,400 Xg) for 2 min, and the solution is collected into a centrifuge tube to obtain seed potato sample DNA.
EXAMPLE 2 DNA extraction of bacterial wilt samples
1. Taking 1-5 mL of bacterial culture solution of Ralstonia solanacearum, centrifuging at 10,000 rpm (11,500 Xg) for 1min, and sucking the supernatant as much as possible.
2. Adding 200 mu L buffer GA into the thallus sediment, and oscillating until the thallus is thoroughly suspended.
3. 20 mu L of protease K solution is added into the tube and uniformly mixed.
4. 220 mu L buffer GB is added, the mixture is oscillated for 15 sec, the mixture is placed at 70 ℃ for 10 min, the solution is clear in strain, and the mixture is centrifuged briefly to remove water drops on the inner wall of the tube cover. ( Note that: white precipitate may be generated when buffer GB is added, and generally disappears when the buffer GB is placed at 70 ℃, so that subsequent experiments are not affected. If the solution is not clear, indicating that the cell lysis is not complete, it may result in a small amount of extracted DNA and an impure extracted DNA. )
5. Adding 220 mu L absolute ethyl alcohol, fully oscillating and uniformly mixing for 15 sec, at this time, possibly generating flocculent precipitate, and briefly centrifuging to remove water drops on the inner wall of the pipe cover.
6. The solution obtained in the previous step and the flocculent precipitate were both put into an adsorption column CB3 (the adsorption column was placed in a collection tube) and centrifuged at 12,000rpm (13,400 Xg) for 30 sec, and the waste liquid was poured off, and the adsorption column CB3 was placed in the collection tube.
7. 500 [ mu ] L buffer GD (absolute ethyl alcohol is added before use, the volume is added with reference to the label on the bottle) is added to the adsorption column CB3, centrifugation is carried out for 30 sec at 12,000rpm (13,400 Xg), waste liquid is poured out, and the adsorption column CB3 is placed in a collecting pipe.
8. 600 μl of rinse solution PW (absolute ethanol is added before use, the volume is added with reference to the label on the bottle) is added to the adsorption column CB3, the solution is centrifuged at 12,000rpm (13,400X g) for 30 sec, the waste liquid is poured off, and the adsorption column CB3 is placed in a collection tube.
9. The operation 8 is repeated.
10. The adsorption column CB3 was put back into the collection tube and centrifuged at 12,000rpm (13,400 Xg) for 2 min, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material. Note that: the purpose of this step is to remove the residual rinse solution from the column, which residual ethanol can affect subsequent enzymatic reactions (digestion, PCR, etc.).
11. Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2-5 min, centrifuging at 12,000rpm (13,400 Xg) for 2 min, and collecting the solution into the centrifuge tube to obtain the bacterial wilt sample DNA. Note that: the volume of the eluting buffer solution is not less than 50 mu L, and the recovery efficiency is affected by the too small volume. The pH of the eluate has a great influence on the elution efficiency. If ddH is used 2 O is used as eluent to ensure that the pH value is in the range of 7.0-8.5, the pH value is lower than 7.0, the eluting efficiency is reduced, and the DNA product is kept at-20 ℃ to prevent DNA degradation. To increase the yield of genomic DNA, the solution obtained by centrifugation may be added to an adsorption column CB3, left at room temperature for 2 min, and centrifuged at 12,000rpm (13,400 Xg) for 2 min.
Example 3 primer set design
The secretion system regulated gene hrpB (NCBI GenBank database, 69784146) with the bacterial wilt type III, and the bacterial wilt flagellin gene flic (NCBI GenBank database, AF 283285.1). LAMP Primer design was performed using Primer Explorer V5 software (http:// Primer Explorer. Jp/LAMP), followed by optimal screening, with each gene obtaining a set of preferred primers. Each set includes an outer forward primer sequence (F3), an outer reverse primer sequence (B3), an inner forward primer sequence (FIP), an inner reverse primer sequence (BIP), and two loop primer sequences (LF and LB), as shown in Table 1, table 1 and Table 2 primers were tested in the laboratory and in the field.
TABLE 1 hrpB LAMP detection primers
TABLE 2 flic LAMP detection primers
EXAMPLE 4 Loop-mediated isothermal amplification
Preparing a reaction system: LAMP reaction systems are prepared, including DNA templates, primer mixtures, LAMP enzymes (e.g., bst DNA polymerase), buffers, and other auxiliary reagents. Ensure that the reaction system is prepared and optimized according to the instruction provided by the manufacturer, and then the optimal system is selected.
Specifically, the DNA of a potato bacterial wilt standard strain UW551 is used as a template to optimize an LAMP reaction system, and the system comprises: 2.5 Mu L10 x ThermoPol buffer, 1.5 mu L MgSO 4 (100 mM), 3.5. Mu.L dNTPs (2.5 mM), 1. Mu.L each of inner primer FIP/BIP (10. Mu.M), 0.5. Mu.L each of outer primer F3/B3 (10. Mu.M), 1. Mu.L each of loop primer LF/LB (10. Mu.M), 1. Mu. L Bst DNA Polymerase solution (320 U.ml), 1. Mu.L template DNA, make up ddH 2 O to 25. Mu.L. The LAMP reaction was carried out in a heating module at a reaction temperature of 63℃for a reaction time of 60 min.
EXAMPLE 5 experiments with the primer set of the present invention
To contain the main disease (early blight) in potatoAlternaria alternataEarly blight and epidemic diseaseAlternaria solaniEpidemic disease in late stagePhytophthora infestansColi, E.coliEnterobacter spBlack moleRhizoctonia solaniDry rot disease of the human bodyFusarium oxysporumAnthracnose of a patientColletotrichum coccodes) The potato sample to be detected is used as a control, and the laboratory detection and the field detection are simultaneously carried out, and the detection method comprises the following steps:
and (3) detecting in a laboratory: the amplified products were subjected to 2% agarose gel electrophoresis. If no target amplification strip appears in the electrophoresis result, judging that the sample to be detected does not contain the bacterial wilt; if the target amplification band appears in the electrophoresis result, the sample to be detected is judged to contain the bacterial wilt.
And (3) field detection: and (3) covering the tube cover tightly by 1 mu L of color reagent (1000 XSYBR Green I) at the central position of the end cover of the top end of the reaction tube. After the reaction, the color developing agent at the top of the cap was mixed upside down to observe the color change. The amplified product was orange, indicating no presence of ralstonia solanacearum. The amplified product was green, indicating that there was bacterial wilt.
As shown in the figure 1, the detection result shows that the primer shows positive effect only when the potato is infected with the bacterial wilt, but not when the potato is infected with other main diseases of the potato, so that the primer has good specificity and can be applied to field detection of the bacterial wilt.
Example 6 sensitivity test of the primer set of the present invention
Serial 10-fold dilutions were performed using standard strain UW551 DNA as template to give concentrations of 100X 100 ng/. Mu.L and 100X 10, respectively -1 ng/μL、100×10 -2 ng/μL、100×10 -3 ng/μL、100×10 -4 ng/μL、100×10 -5 ng/μL、100×10 -6 ng/. Mu.L was used to verify LAMP sensitivity. The detection result is detected by agarose gel electrophoresis, and can also be observed by SYBR Green I staining. Meanwhile, the DNA with different concentrations is amplified by using a bacterial wilt PCR specific primer Au759F/Au760R, and the amplified DNA band size is 280bp. Comparison of LAMP and PCR sensitivity. As shown in FIG. 2, the sensitivity of the nucleic acid primer set for LAMP amplification of the present invention was 100 times that of the conventional PCR, which is a conventional and main detection method for detecting bacterial wilt in a laboratory, and it was found that it took a long time and only half of the time was required for LAMP detection.
EXAMPLE 7 experiments on the effect of the application of the primer set of the present invention
As shown in fig. 3, lanes 1, 4, 5, 6, 7, 9 and 10 are potatoes without bacterial wilt, lanes 2, 3 and 8 are potatoes with bacterial wilt, and the laboratory detection and field detection results are consistent.
The invention also carries out research on specificity, sensitivity and application effect by using the flic LAMP detection primer, and experimental results are the same as those of the hrpB LAMP detection primer, so that the primer group has strong specificity and high sensitivity when being used for detecting the bacterial wilt and can meet the field detection.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
Claims (10)
1. A nucleic acid primer set for detecting bacterial wilt, characterized in that the nucleic acid primer set comprises any one of a primer set i for amplifying a bacterial wilt type iii secretion system regulatory gene hrpB and a primer set ii for amplifying a bacterial wilt flagellin gene flic;
the primer group I comprises F3 with nucleotide sequences shown as SEQ ID NO. 1-6 respectively i Primer, B3 i Primers, FIP i Primer, BIP i Primer, LF i Primers and LB i A primer;
the primer group II comprises F3 with nucleotide sequences shown as SEQ ID NO 7-12 respectively j Primer, B3 j Primers, FIP j Primer, BIP j Primer, LF j Primers and LB j And (5) a primer.
2. A loop-mediated isothermal amplification reagent comprising the nucleic acid primer set according to claim 1.
3. A kit for detecting ralstonia solanacearum, comprising the loop-mediated isothermal amplification reagent of claim 2.
4. Use of the nucleic acid primer set of claim 1 or the reagent of claim 2 or the kit of claim 3 for detecting ralstonia solanacearum.
5. Use of the nucleic acid primer set of claim 1 or the reagent of claim 2 or the kit of claim 3 for detecting ralstonia solanacearum.
6. A method of detecting ralstonia solanacearum, the method comprising:
extracting DNA of a sample to be detected;
performing loop-mediated isothermal amplification on the DNA of the sample to be detected by adopting the nucleic acid primer set of claim 1 to obtain an amplification product;
detecting the amplified product; the method comprises the steps of,
and judging whether the sample to be detected contains the bacterial wilt or not based on the detection result.
7. The method of claim 6, wherein the loop-mediated isothermal amplification reaction system comprises: 2.5 Mu L10 XThermoPol buffer, 1.5 mu L MgSO 4 、3.5 μL dNTPs、FIP i Primers or FIP j Primer 1 mu L, BIP i Primers or BIP j Primer 1 mu L, F3 i Primers or F3 j Primer 0.5 mu L, B3 i Primer or B3 j Primer 0.5 mu L, LF i Primers or LF j Primer 1 mu L, LB i Primers or LB j 1. Mu.L of primer, 1. Mu. L Bst DNA Polymerase solution, 1. Mu.L of template DNA, make up ddH 2 O to 25. Mu.L.
8. The method of claim 6, wherein the loop-mediated isothermal amplification procedure comprises: and (3) performing amplification reaction at the constant temperature, wherein the reaction temperature is 60-65 ℃ and the reaction time is 50-70 min.
9. The method of claim 6, wherein the detection is performed by agarose gel electrophoresis of the amplified product, and the determination criteria are: if no target amplification strip appears in the electrophoresis result, judging that the sample to be detected does not contain the bacterial wilt; if the target amplification band appears in the electrophoresis result, the sample to be detected is judged to contain the bacterial wilt.
10. The method of claim 6, wherein the detection is performed by contacting the amplified product with a color reagent 1000 x SYBR Green I to react, and the criterion is: if the reaction liquid is orange, judging that the sample to be detected does not contain the bacterial wilt; if the reaction liquid is green, the sample to be detected is judged to contain the bacterial wilt.
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