Kit for detecting alfalfa rhizoctonia solani and detection method thereof

Abstract

The invention discloses a kit for detecting rhizoctonia solani of alfalfa and a detection method thereof, and relates to the technical field of biology. The detection kit and the detection method disclosed by the invention have the characteristic of strong specificity, can quickly and accurately detect whether alfalfa is infected with the alfalfa rhizoctonia solani or not, and can distinguish the alfalfa from other common germs such as phytophthora root rot germs; meanwhile, the detection kit and the detection method disclosed by the invention can identify whether the alfalfa rhizoctonia solani exists or not from a small amount of alfalfa samples, and have the characteristic of high sensitivity; the PCR primer combination for detecting the alfalfa rhizoctonia solani also has the characteristics of short time consumption and simplicity and convenience in operation, and the detection can be completed in a relatively short time under laboratory conditions.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a kit for detecting Rhizoctonia solani and a detection method thereof. Background Art

[0002] Alfalfa (Medicago sativa, English name Alfalfa) is famous for its high crude protein content, good palatability, rich nutrition and other characteristics. It is a high-quality feed required by livestock and is known as the "first workshop for high-quality milk production." In addition, alfalfa has a well-developed root system and a strong nitrogen fixation ability. It can be rotated with other crops, which helps to optimize the structure and function of the farmland ecosystem, thus having a certain ecological value. However, in recent years, the continuous extension of the utilization period of alfalfa and the gradual expansion of the planting area have led to diseases becoming an important factor restricting the development of the alfalfa industry, which has had a great negative impact on the yield and quality of alfalfa.

[0003] Alfalfa root rot is common in alfalfa-growing areas around the world. The disease mainly causes the rot of alfalfa roots and root crowns, making it very difficult for the roots to absorb water and nutrients, greatly reducing the nitrogen fixation capacity of alfalfa. In severe cases, it can lead to plant death, resulting in a serious decline in alfalfa yield and quality. Among them, Rhizoctonia solani is also an important type of pathogen that causes alfalfa root rot. It is a soil-borne pathogenic fungus with a wide host range, strong saprophytic properties, and widespread distribution throughout the world. Once alfalfa is infected by the fungus, symptoms such as root rot and leaf blight will occur. Therefore, a quick and accurate quarantine method is crucial to prevent the fungus from harming the growth of alfalfa on a large scale.

[0004] Existing detection methods lack a rapid and accurate method for targeting Rhizoctonia solani in alfalfa.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a kit for detecting Rhizoctonia solani and a detection method thereof. The kit and the detection method of the present invention can detect Rhizoctonia solani quickly, efficiently and accurately.

[0007] In a first aspect, the present invention provides a detection primer for Rhizoctonia solani of alfalfa, comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.2.

[0008] In a second aspect, the present invention provides the use of the above detection primers in detecting Rhizoctonia solani.

[0009] In a third aspect, the present invention provides use of the above detection primers in preparing a kit for detecting Rhizoctonia solani.

[0010] In a fourth aspect, the present invention provides a kit for detecting Rhizoctonia solani, comprising the above-mentioned detection primers.

[0011] In a fifth aspect, the present invention also provides the use of the above-mentioned kit in detecting Rhizoctonia solani.

[0012] In a sixth aspect, the present invention further provides a method for detecting Rhizoctonia solani, comprising:

[0013] Using the DNA of the sample to be tested as an amplification template and the above detection primers, a PCR reaction system is established to perform PCR amplification;

[0014] The amplified product is detected. If the target band is amplified, it is determined that the sample to be tested contains Rhizoctonia solani; otherwise, it is determined that the sample to be tested does not contain Rhizoctonia solani.

[0015] The present invention has the following beneficial effects:

[0016] (1) The present invention can amplify a single band for the genomic DNA of Rhizoctonia solani, has strong specificity, can quickly and accurately detect whether alfalfa is infected with Rhizoctonia solani, and can distinguish it from other common pathogens such as Phytophthora root rot fungi;

[0017] (2) The detection kit and the detection method of the present invention can identify whether Rhizoctonia solani is present in a small amount of alfalfa samples, and have the characteristic of high sensitivity;

[0018] (3) The detection of Rhizoctonia solani using the PCR primer combination of the present invention is also time-saving and easy to operate, and the detection can be completed in a short time under laboratory conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 The PCR identification results of whether 15 alfalfa seed varieties contain Rhizoctonia solani;

[0021] Figure 2 To identify the sensitivity of the Rhizoctonia solani kit in different numbers of seeds of Alfalfa King, Suntory, Gannong No. 1 and Gongnong No. 1, the PCR results;

[0022] Figure 3 for Figure 1 Sequencing and comparison of PCR products of 40 seeds of Zhongmu 6 and Zhonglan 1;

[0023] Figure 4 The specific identification results of PCR primers for detecting Rhizoctonia solani of alfalfa;

[0024] Figure 5 PCR results of using different primers to identify whether 15 alfalfa seed varieties contain Rhizoctonia solani. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] In order to quickly and accurately detect the colonization of Rhizoctonia solani in alfalfa seeds, the present invention designs a pair of PCR detection primers for Rhizoctonia solani, and the primer pair is:

[0027] MsRis-F(5′-3′):ATGCCTCGAAGAATCCCTGAT-SEQ ID NO.1;

[0028] MsRis-R(5'-3'): TTAAGATTGTACGGGTAACATGTTAAAT-SEQ ID NO. 2.

[0029] Based on the above primer pair, the present invention can provide a detection kit for Rhizoctonia solani of alfalfa, which comprises the above primer pair and other conventional reagents for PCR amplification, including PCR amplification reagents, DNA templates and ddH2O.

[0030] Among them, the PCR amplification reagent includes Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, and other commonly used auxiliary reagents.

[0031] In addition to the PCR amplification reagent provided by the present invention, the PCR reaction system can also use commercial products of the same type, and PCR amplification can be performed by adding a specified amount of DNA template and primers before amplification. The premise is that the positive control sample must amplify the correct band, and the negative control sample has no amplified band.

[0032] By amplifying the DNA of the test samples containing or not containing Rhizoctonia solani, it was found that the target band could be amplified from the test samples only in the presence of Rhizoctonia solani.

[0033] Since the above detection primers or kit can be used to identify whether an alfalfa sample contains Rhizoctonia solani, the present invention also provides a method for detecting whether an alfalfa sample contains Rhizoctonia solani, wherein the sample to be tested can be an alfalfa seed, or an alfalfa root, a leaf, or other tissue.

[0034] Taking alfalfa seeds as an example, the method for detecting whether alfalfa seeds contain Rhizoctonia solani includes the following steps:

[0035] (1) Extracting genomic DNA from alfalfa seeds to be tested;

[0036] (2) using the DNA obtained in step (1) as a template and the above primer pair to perform a PCR amplification reaction;

[0037] (3) taking the PCR amplification product and detecting it by agarose gel electrophoresis;

[0038] (4) judging whether the alfalfa seeds to be tested are colonized with Rhizoctonia solani based on whether characteristic bands appear in the electrophoresis bands: if bands appear in the electrophoresis results, the alfalfa seeds are colonized with Rhizoctonia solani; if no bands appear, the alfalfa seeds are not colonized with Rhizoctonia solani.

[0039] In the above detection method, the reaction system of PCR amplification is: 12.5 μL of PCR amplification reagent, 1.0 μL of upstream primer, 1.0 μL of downstream primer, 1 μL of template DNA, 9.5 μL of ddH2O; wherein the concentration of the DNA template is 0.1-5 μg / mL; and the concentration of the upstream primer and the downstream primer is 10 μM.

[0040] The reaction program of PCR amplification was as follows: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 30 s, annealing at 58°C for 10 s, extension at 72°C for 5 s, 35 cycles, and extension at 72°C for 1 min.

[0041] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0042] Example 1

[0043] This embodiment is a kit for detecting Rhizoctonia solani of alfalfa, and its components include: a PCR amplification primer pair, 2×Phanta Flash Master Mix (Dye Plus) (purchased from Novozymes Biotech), a DNA template and ddH2O.

[0044] Among them, the PCR amplification primer pair is:

[0045] MsRis-F(5′-3′):ATGCCTCGAAGAATCCCTGAT-SEQ ID NO.1;

[0046] MsRis-R(5'-3'): TTAAGATTGTACGGGTAACATGTTAAAT-SEQ ID NO. 2.

[0047] Example 2

[0048] This embodiment is a method for detecting whether alfalfa seeds are colonized with Rhizoctonia solani, which is performed using the detection kit in Example 1, and the specific steps are as follows:

[0049] S1. Collect alfalfa seeds of different varieties and extract genomic DNA for later use.

[0050] S2. Using the PCR amplification primer pair in Example 1, perform PCR detection on the seed genomic DNA.

[0051] The total volume of the PCR detection system was 25 μL, including: 12.5 μL of 2×Phanta Flash Master Mix (Dye Plus), 1 μL of upstream primer, 1 μL of downstream primer; 1 μL of DNA template (concentration less than 600 ng / μL); 9.5 μL of ddH2O.

[0052] Amplification conditions were as follows: (1) pre-denaturation at 98°C for 30 s; (2) denaturation at 98°C for 30 s; annealing at 58°C for 10 s; (4) extension at 72°C for 5 s; (5) (2) to (4) steps were cycled 35 times; (6) extension at 72°C for 1 min;

[0053] S3. Use 1.5% agarose gel for detection, weigh 1.5g agarose, add 100mL of prepared 1×TAE, heat in a microwave oven for about 2min until the agarose is completely dissolved, add 5μL of nucleic acid dye and shake well, then pour the agarose solution into the gel mold, solidify for 1h, run the gel at 140V for 20min in a vertical plate electrophoresis apparatus, and observe the results under a gel imager. Repeat the above at least three times.

[0054] Test results such as Figure 1As shown in the figure, M represents Marker; CK+ represents positive control (Rhizoctonia solani); CK- represents negative control (water); 1-15 are genomic DNA extracted from seeds of different varieties of alfalfa shown in Table 1. All genomic DNAs were extracted three times independently and PCR assays were performed respectively. The gel image is a random image of three biological replicates.

[0055] Table 1 Information on the 15 participating alfalfa varieties

[0056]

[0057] from Figure 1 As can be seen, bands were detected in the seeds of alfalfa varieties 2-3 and 5-7, but no bands were detected in the other 10 varieties.

[0058] right Figure 1 The PCR products with detected bands were sequenced and analyzed. Specifically, the PCR products of 40 seeds of Zhongmu 6 and Zhonglan 1 were sequenced and compared. The results are as follows Figure 2 As shown in the figure, the red parts at the beginning and end of the common sequence represent the special primers MsRis-F and MsRis-R for identifying whether alfalfa seeds contain Rhizoctonia solani, and the sequencing results have a similarity of up to 99% with the template sequence.

[0059] Experimental Example 1

[0060] This experiment is a sensitivity detection test of the kit in Example 1, and is specifically as follows:

[0061] S1. The genomic DNA was re-extracted from 1, 10, 20, 40 and 60 alfalfa seeds of the varieties detected to contain Rhizoctonia solani in Example 2 (American Alfalfa King, Suntory, Gannong No. 1 and Gongnong No. 1) for PCR detection.

[0062] The total volume of the PCR detection system was 25 μL, including: 12.5 μL of 2×Phanta Flash Master Mix (Dye Plus), 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of DNA template, and 9.5 μL of ddH2O.

[0063] Among them, in the DNA template: the concentration of 1 seed is 153ng / μL; the concentration of 10 seeds is 191ng / μL; the concentration of 20 seeds is 305ng / μL; the concentration of 40 seeds is 387ng / μL; the concentration of 60 seeds is 443ng / μL.

[0064] The amplification conditions were as follows: (1) pre-denaturation at 98°C for 30 s; (2) denaturation at 98°C for 30 s; annealing at 58°C for 10 s; (4) extension at 72°C for 5 s; (5) steps (2) to (4) were cycled 35 times; and (6) extension at 72°C for 1 min.

[0065] S2. Use 1.5% agarose gel for detection, weigh 1.5g agarose, add 100mL of prepared 1×TAE, heat in a microwave oven for about 2min until the agarose is completely dissolved, add 5μL of nucleic acid dye and shake well, then pour the agarose solution into the gel mold, solidify for 1h, run the gel at 140V for 20min in a vertical plate electrophoresis instrument, and then observe the results under a gel imager.

[0066] Test results such as Figure 3 As shown in Figures A, B, C and D, No. 1-5 represent the PCR identification results of genomic DNA extracted from 60, 40, 20, 10 and 1 seeds of alfalfa seeds of American Alfalfa King, Suntory, Gannong No. 1 and Gongnong No. 1, respectively; CK+ represents the positive control (Rhizoctonia solani); CK- represents the negative control (water); M represents Marker. All genomic DNA was extracted three times independently and PCR assayed respectively. The gel image is a random image of three biological replicates.

[0067] Figure 3 It can be seen that bands can be detected in the genomic DNA of 60 seeds, 40 seeds, 20 seeds, and 10 seeds of American Alfalfa King, Suntory, and Gannong No. 1, while no bands can be detected in the genomic DNA of 60 seeds, 40 seeds, 20 seeds, and 10 seeds of Gongnong No. 1.

[0068] Experimental Example 2

[0069] This experiment is a specificity verification test of the kit in Example 1, and is specifically as follows:

[0070] S1. Extract genomic DNA of Rhizoctonia solani and Phytophthora root rot pathogens respectively for later use;

[0071] S2. Using the PCR amplification primer pair in Example 1, PCR detection was performed on the genomic DNA of Rhizoctonia solani and Phytophthora root rot pathogens.

[0072] The total volume of the PCR detection system is 25 μL, including 12.5 μL of 2×Phanta Flash Master Mix (Dye Plus), 1 μL of upstream primer, 1 μL of downstream primer; DNA template (concentration less than 600 ng / μL): 1 μL; 9.5 μL of ddH2O.

[0073] The amplification conditions were as follows: (1) pre-denaturation at 98°C for 30 s; (2) denaturation at 98°C for 30 s; annealing at 58°C for 10 s; (4) extension at 72°C for 5 s; (5) steps (2) to (4) were cycled 35 times; and (6) extension at 72°C for 1 min.

[0074] S3. Use 1.5% agarose gel for detection, weigh 1.5g agarose, add 100mL of prepared 1×TAE, heat in a microwave oven for about 2min until the agarose is completely dissolved, add 5μL of nucleic acid dye and shake well, then pour the agarose solution into the gel mold, solidify for 1h, run the gel at 140V for 20min in a vertical plate electrophoresis instrument, and then observe the results under a gel imager.

[0075] Test results such as Figure 4 As shown, M represents Marker; CK+ represents positive control (Rhizoctonia solani); CK- represents negative control (water); 1 represents Rhizoctonia solani; 2-4 represents Phytophthora root rot pathogen. All genomic DNA was extracted three times independently and PCR assayed respectively. The gel image is a random image from three biological replicates.

[0076] from Figure 4 It can be seen that there are no bands in lanes 2-4, indicating that the detection primers of the present invention can only specifically identify samples containing Rhizoctonia solani.

[0077] Experimental Example 3

[0078] This experiment used existing Rhizoctonia solani detection primers to perform PCR detection on genomic DNA of different varieties of alfalfa seeds, and compared the results with the results in Example 2 to test the sensitivity of the detection primers in the present invention. The specific information of the existing Rhizoctonia solani detection primers is shown in Table 2:

[0079] Table 2 Information on existing primers for Rhizoctonia solani detection

[0080]

[0081] The above existing detection primers were used to identify the 15 alfalfa seed varieties in Table 1. The test results are as follows: Figure 5 As shown in the figure: A and B represent the PCR results of identifying whether 15 alfalfa seed varieties contain Rhizoctonia solani using the authorized patent primers 1 and 2; where M is a marker; CK+ is a positive control (Rhizoctonia solani); CK- is a negative control (water); 1-15 are DNA extracted from seeds of different alfalfa varieties shown in Table 1. All genomic DNA was extracted three times independently and PCR assayed respectively. The gel image is a random image of three biological replicates.

[0082] from Figure 5 It can be seen that the existing primers for detecting Rhizoctonia solani cannot identify whether alfalfa seeds No. 1-15 contain Rhizoctonia solani.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A detection primer for Rhizoctonia solani, characterized in that: It comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.

2.

2. Use of the detection primer as claimed in claim 1 in detecting Rhizoctonia solani.

3. Use of the detection primer as claimed in claim 1 in preparing a kit for detecting Rhizoctonia solani.

4. A kit for detecting Rhizoctonia solani, characterized in that: Comprising the detection primer described in claim 1.

5. The kit according to claim 4, characterized in that The kit also includes PCR amplification reagents, DNA templates and ddH2O.

6. The kit according to claim 5, characterized in that The PCR amplification reagent includes Taq DNA polymerase, dNTPs, MgCl2 and reaction buffer.

7. Use of the kit according to any one of claims 4 to 6 in detecting Rhizoctonia solani.

8. A method for detecting Rhizoctonia solani, characterized in that: include: Using the DNA of the sample to be tested as an amplification template, using the detection primers described in claim 1, establishing a PCR reaction system, and performing PCR amplification; The amplified product is detected. If the target band is amplified, it is determined that the sample to be tested contains Rhizoctonia solani; otherwise, it is determined that the sample to be tested does not contain Rhizoctonia solani.

9. The detection method according to claim 8, characterized in that: The reaction system is: 12.5 μL of PCR amplification reagent, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of template DNA, and 9.5 μL of ddH2O; The concentration of the upstream primer and the downstream primer is 10 μM; the concentration of the template DNA is 0.1-5 μg / mL.

10. The detection method according to claim 8, characterized in that: The reaction program of PCR amplification was as follows: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 30 s, annealing at 58°C for 10 s, extension at 72°C for 5 s, 35 cycles, and extension at 72°C for 1 min.

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