Kit for detecting alfalfa seed bees and detection method thereof

Abstract

The invention discloses a kit for detecting alfalfa seed bees and a detection method thereof, and relates to the technical field of biology. The PCR primer combination for detecting the clover eggs can effectively identify whether clover eggs are mixed in clover seeds or not, and the method for identifying the clover eggs by using the primers has the characteristics of strong sensitivity, high accuracy, strong repeatability and the like. Meanwhile, when the PCR primer combination is used for detecting the alfalfa seed bee eggs, large-batch detection of the alfalfa seed bee eggs can be achieved within a short time, operation is easy, and cost is low. Therefore, the detection primer and the detection method provided by the invention have good application prospects.

Description

Technical Field

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

[0002] Alfalfa (Medicago sativa) is a widely planted forage crop with high nutritional value and strong adaptability. It is an indispensable and important feed source in the global animal husbandry industry. However, during the planting and seed production process of alfalfa, it is often threatened by pests and diseases, among which the alfalfa seed bee (Bruchophagus gibbus) is particularly serious. The alfalfa seed bee lays eggs in the seeds during seed maturation, causing damage to the seeds, which in turn seriously damages the quality and yield of the seeds, posing a major challenge to the development of the alfalfa industry.

[0003] Currently, one of the main constraints in alfalfa seed production is low seed yield, which is largely attributed to the infestation of pests such as alfalfa seed bees. Traditionally, pest detection of alfalfa seeds relies on manual screening and visual inspection, which is not only inefficient but also prone to missed detections, and cannot guarantee the purity and quality of the seeds. Therefore, developing an efficient and accurate method for identifying alfalfa seed bee eggs is of great significance for improving the production efficiency and quality of alfalfa seeds.

[0004] With the development of science and technology, especially the continuous progress of molecular biology and detection technology, new ideas and methods have been provided for pest identification. However, there is no widely recognized standard for the specific identification method of alfalfa seed bee eggs, which limits the effective prevention and control of pests in alfalfa seed production.

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

[0006] The purpose of the present invention is to provide a kit for detecting alfalfa bee and a detection method thereof. The kit and the detection method of the present invention can detect alfalfa bee eggs quickly, efficiently and accurately.

[0007] In a first aspect, the present invention provides a PCR primer combination for detecting alfalfa seed bee insect eggs, comprising: a forward primer as shown in SEQ ID NO.1, and a reverse primer as shown in SEQ ID NO.2.

[0008] In a second aspect, the present invention provides the use of the above-mentioned PCR primer combination in the preparation of a kit for detecting alfalfa seed bee insect eggs.

[0009] In a third aspect, the present invention provides a kit for detecting alfalfa seed bee insect eggs, which comprises the above-mentioned PCR primer combination.

[0010] In a fourth aspect, the present invention provides the use of the above-mentioned kit in detecting alfalfa seed bee insect eggs.

[0011] In a fifth aspect, the present invention further provides a method for detecting whether alfalfa seeds contain alfalfa bee insect eggs, comprising:

[0012] Extracting genomic DNA from alfalfa seeds, then using the extracted DNA as a template, performing PCR amplification using the kit described in the claim, and then performing agarose gel electrophoresis on the PCR product;

[0013] If bands appear in the electrophoresis results, the alfalfa seeds contain alfalfa seed bee worm eggs; if no bands appear, the alfalfa seeds do not contain alfalfa seed bee worm eggs.

[0014] The present invention has the following beneficial effects:

[0015] (1) The PCR primer combination for detecting alfalfa seed bee worm eggs designed by the present invention can effectively identify whether alfalfa seeds are mixed with alfalfa seed bee worm eggs. The method for identifying alfalfa seed bee worm eggs using the primers has the characteristics of high sensitivity, high accuracy, high repeatability, etc.;

[0016] (2) The PCR primer combination of the present invention can be used to detect alfalfa seed bee eggs, and a large number of alfalfa seeds can be detected at the same time. The operation is simple and the detection can be completed in a short time under laboratory conditions.

[0017] (3) The detection method provided by the present invention is applicable to a variety of alfalfa varieties and has a strong promotion value. It is helpful for alfalfa seed inspectors to detect whether alfalfa seeds are mixed with alfalfa bee and insect eggs, so that alfalfa seeds can better serve the livestock production and lay a solid foundation for ensuring my country's food security. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 This is the relevant information about alfalfa seed bees. Figure A shows the destructiveness of alfalfa seed bees to alfalfa seeds; Figure B is a real photo of alfalfa seed bees;

[0020] Figure 2 The PCR identification results of 14 alfalfa varieties and 5 common alfalfa pests for the presence of alfalfa seed bee;

[0021] Figure 3 To identify the PCR identification results of the sensitivity of the alfalfa seed bee kit for different numbers of seeds of Zhongtian No. 1, Xinjiang Daye, Suntory, and Jinneng 5010;

[0022] Figure 4 for Figure 3 Sequencing and comparison results of PCR products from 40 seeds of Suntory and Jinneng 5010. DETAILED DESCRIPTION

[0023] 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.

[0024] Alfalfa seed bees belong to the family Mylidae, and they mainly live in plants such as purple alfalfa, blue alfalfa, yellow alfalfa, red clover, white clover, and sweet clover. Alfalfa seed bees lay eggs in seeds during the seed maturation period, causing damage to the seeds, which in turn seriously damages the quality and yield of the seeds. Figure 1 As shown, 1A shows the destructiveness of alfalfa seed bees to alfalfa seeds, and 1B is a real photo of alfalfa seed bees.

[0025] In order to overcome the problems of low efficiency and easy missed detection in manual screening and visual inspection, the present invention designs a PCR detection primer pair for alfalfa seed bee insect eggs, and the primer pair is:

[0026] MBNEL-F(5′-3′):GATAGCGAACAAGTACCGTGAG-SEQ ID NO.1;

[0027] MBNEL-R(5′-3′):CGTCCTACTAGGGGAGAAGTG-SEQ ID NO.2.

[0028] Based on the above primer pair, the present invention can provide a detection kit for alfalfa seed bee insect eggs, which contains the above primer pair and other conventional reagents for PCR amplification, including PCR amplification reagents, DNA templates and ddH2O.

[0029] The PCR amplification reagent includes Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, and conventional PCR reaction enhancers, optimizers and stabilizers.

[0030] 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.

[0031] By amplifying the DNA of alfalfa seeds with or without alfalfa seed bee eggs, it was found that the target band could be amplified in the test sample only when alfalfa seed bee eggs were present.

[0032] Since the above detection primers or kits can be used to identify whether alfalfa seeds contain alfalfa bee insect eggs, the present invention also provides a method for detecting whether alfalfa seeds contain alfalfa bee insect eggs, which method comprises:

[0033] (1) Extracting genomic DNA from alfalfa seeds to be tested;

[0034] (2) using the DNA obtained in step (1) as a template and the above-mentioned primer pair to perform a PCR amplification reaction;

[0035] (3) taking the PCR amplification product and detecting it by agarose gel electrophoresis;

[0036] (4) judging whether the alfalfa seeds to be tested contain alfalfa bee worm eggs according to whether characteristic bands appear in the electrophoresis bands: if bands appear in the electrophoresis results, the alfalfa seeds contain alfalfa bee worm eggs; if no bands appear, the alfalfa seeds do not contain alfalfa bee worm eggs.

[0037] In the above detection method, the reaction system of PCR amplification is: 10 μL of PCR amplification reagent, 0.5 μL of forward primer, 0.5 μL of reverse primer, 0.5 μL of template DNA, 8.5 μL of ddH2O; wherein the concentration of the forward primer and the reverse primer is 10 μmol / L; and the concentration of the template DNA is <600 μg / mL.

[0038] The reaction program of PCR amplification was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 35 cycles, and extension at 72°C for 5 min.

[0039] 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.

[0040] Example 1

[0041] This embodiment is a kit for detecting alfalfa seed bee insect eggs, and its components include: PCR amplification primer pair, 2×PCR MasterMix (purchased from Tiangen Biochemical Technology Co., Ltd.), DNA template and ddH2O.

[0042] Among them, the PCR amplification primer pair is:

[0043] MBNEL-F(5′-3′):GATAGCGAACAAGTACCGTGAG-SEQ ID NO.1;

[0044] MBNEL-R(5′-3′):CGTCCTACTAGGGGAGAAGTG-SEQ ID NO.2.

[0045] Example 2

[0046] This embodiment is a method for detecting whether alfalfa seeds contain alfalfa bee eggs, which is detected using the detection kit in Example 1, and the specific steps are as follows:

[0047] S1. Germinate alfalfa seeds and extract leaf genomic DNA for later use;

[0048] S2. Collect alfalfa seeds and extract genomic DNA for later use;

[0049] S3. Using the primers specifically for alfalfa seeds in Example 1, perform PCR detection on the genomic DNA of leaves and seeds respectively.

[0050] The total volume of the PCR detection system was 20 μL, including 10 μL of 2×PCR MasterMix II, 0.5 μL of DNA template (concentration < 600 ng / μL), 0.5 μL of primer MBNEL-F (concentration 10 μmol / L), 0.5 μL of primer MBNEL-R (concentration 10 μmol / L), and 8.5 μL of ddH2O.

[0051] The reaction procedure of PCR detection is as follows: (1) pre-denaturation at 94°C for 3 min; (2) denaturation at 94°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 30 s; (5) (2) to (4) steps are cycled 35 times; (6) extension at 72°C for 5 min;

[0052] S4. Detection was performed using 1.4% agarose gel, 100 mL system, 98 mL H2O, 2 mL 5×TAE, 1.4 g agarose, heated for 3 min, 10 mL nucleic acid dye was added, solidified for 50 min, run the gel at 140 V for 20 min in a vertical plate electrophoresis instrument, and photographed on a gel imager. PCR products were detected by 8% APEG gel electrophoresis.

[0053] Experimental Example 1

[0054] This experiment is a specificity verification test of the kit in Example 1, and is specifically as follows:

[0055] S1. Germinate different varieties of alfalfa seeds and extract leaf genomic DNA for later use;

[0056] S2. Collect different varieties of alfalfa seeds and extract genomic DNA for later use;

[0057] S3. Using the alfalfa seed-specific primers in Example 1, PCR detection was performed on the genomic DNA of alfalfa leaves and seeds of different varieties and the genomic DNA of other alfalfa pests.

[0058] The total volume of the PCR detection system was 20 μL, including 10 μL of 2×PCR MasterMix II, 0.5 μL of DNA template (concentration < 600 ng / μL), 0.5 μL of primer MBNEL-F (concentration 10 μmol / L), 0.5 μL of primer MBNEL-R (concentration 10 μmol / L), and 8.5 μL of ddH2O.

[0059] The reaction procedure of PCR detection is as follows: (1) pre-denaturation at 94°C for 3 min; (2) denaturation at 94°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 30 s; (5) (2) to (4) steps are cycled 35 times; (6) extension at 72°C for 5 min;

[0060] S4. Use 1.4% agarose gel for detection, 100mL system, 98mL H2O, 2mL 5×TAE, 1.4g agarose, heat for 3min, add 10mL nucleic acid dye, solidify for 50min, run the gel at 140V voltage for 20min in a vertical plate electrophoresis instrument, and take a photo on a gel imager. PCR products were detected by 8% APEG gel electrophoresis, and the results are as follows Figure 2 shown.

[0061] The information of alfalfa varieties and five common alfalfa pests tested in this experiment are shown in Table 1:

[0062] Table 1 Information on 14 alfalfa varieties tested and 5 common alfalfa pests

[0063]

[0064]

[0065] Figure 2In Figures A and B, 1-14 represent genomic DNA from 20 seeds and leaves of different alfalfa varieties; "+" represents alfalfa seed bee (positive control); "-" represents water (negative control); M is a marker; 15-19 represent genomic DNA from common alfalfa parasitic pests; the alfalfa varieties and common alfalfa parasitic pests represented by 1-19 are shown in Table 1. All genomic DNAs were extracted three times independently and PCR assays were performed respectively. The gel images are random images from three biological replicates.

[0066] from Figure 2 It can be seen that among the seeds of alfalfa varieties No. 1-14, bands were detected in 10 types and no bands were detected in 4 types; no bands were detected in the DNA of the leaves; and no bands were detected in common alfalfa pests.

[0067] Experimental Example 2

[0068] This experiment is a sensitivity detection test of the kit in Example 1, and is as follows:

[0069] S1. Re-extract genomic DNA from 1, 10, 20, 40 and 60 alfalfa seeds of Zhongtian No. 1, Xinjiang Daye, Suntory and Jinneng 5010 in Table 1 for PCR detection.

[0070] The total volume of the PCR detection system was 20 μL, including 2×PCR MasterMix II 10 μL, DNA template 0.5 μL, primer MBNEL-F 0.5 μL, primer MBNEL-R 0.5 μL, and ddH2O 8.5 μL. The concentration of the DNA template for 1 seed was 153 ng / μL; the concentration for 10 seeds was 191 ng / uL; the concentration for 20 seeds was 305 ng / μL; the concentration for 40 seeds was 387 ng / μL; and the concentration for 60 seeds was 443 ng / μL.

[0071] The amplification conditions were as follows: (1) pre-denaturation at 94°C for 3 min; (2) denaturation at 94°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 30 s; (5) (2) to (4) steps were cycled 35 times; (6) extension at 72°C for 5 min;

[0072] S2. Use 1.4% agarose gel for detection: 100mL system, 98mL H2O, 2mL 5×TAE, 1.4g agarose, heat for 3min, add 10mL nucleic acid dye, solidify for 50min, run the gel at 140V for 20min in a vertical plate electrophoresis apparatus, and take pictures on a gel imager.

[0073] The results are as follows 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, respectively, from Zhongtian No. 1, Xinjiang Daye, Suntory and Jinneng 5010; "+" represents alfalfa seed bee (positive control); "-" represents water (negative control); M is a marker. All genomic DNA was extracted three times independently and PCR assayed respectively. The gel image is a random image from three biological replicates.

[0074] Figure 3 As can be seen from AB, the genomic DNA of 60, 40, and 20 seeds of Zhongtian No. 1 can detect bands, and the genomic DNA of 60, 40, 20, and 10 seeds of Xinjiang Daye can detect bands; Figure 3 As shown in CD, no bands were detected in the genomic DNA of 60, 40, 20 and 10 seeds of Suntory and Jinneng 5010. According to the above results, it can be concluded that even with a small number of alfalfa seeds as samples, the detection primers of the present invention can detect whether some varieties contain alfalfa seed bees, which also proves that the detection primers of the present invention have high detection sensitivity.

[0075] right Figure 3 The PCR products with detected bands were sequenced and analyzed. Figure 4 As shown, the sequencing results have a similarity of up to 99% with the template sequence.

[0076] 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 PCR primer combination for detecting alfalfa seed bee insect eggs, characterized in that: include: The forward primer is shown as SEQ ID NO.1, and the reverse primer is shown as SEQ ID NO.

2.

2. Use of the PCR primer combination as claimed in claim 1 in preparing a kit for detecting alfalfa seed bee insect eggs.

3. A kit for detecting alfalfa seed bee ova, characterized in that: Comprising the PCR primer combination according to claim 1.

4. The kit according to claim 3, characterized in that The kit also includes PCR amplification reagents, DNA templates and ddH2O.

5. The kit according to claim 4, characterized in that The PCR amplification reagent includes Taq DNA polymerase, dNTPs, MgCl2 and reaction buffer.

6. Use of the kit as described in any one of claims 3 to 5 in detecting alfalfa seed bee insect eggs.

7. The application according to claim 6, characterized in that: The application includes identifying whether alfalfa seeds contain alfalfa seed bee eggs.

8. A method for detecting whether alfalfa seeds contain alfalfa bee eggs, characterized in that: include: Extracting genomic DNA from alfalfa seeds, then using the extracted DNA as a template, performing PCR amplification using the kit described in any one of claims 3 to 5, and then detecting the PCR product by agarose gel electrophoresis; If there are bands in the electrophoresis results, the alfalfa seeds contain alfalfa bee worm eggs; If no bands appear, the alfalfa seeds do not contain alfalfa bee eggs.

9. The method according to claim 8, characterized in that The reaction system for PCR amplification was: 10 μL of PCR amplification reagent, 0.5 μL of forward primer, 0.5 μL of reverse primer, 0.5 μL of template DNA, and 8.5 μL of ddH2O; The concentrations of the forward primer and the reverse primer are 10 μmol / L; the concentration of the template DNA is less than 600 μg / mL.

10. The method according to claim 8, characterized in that The reaction program of PCR amplification was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 35 cycles, and extension at 72°C for 5 min.

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