Kit and detection method for identifying dipsacus aschersonius

Abstract

The invention discloses a kit for detecting alfalfa seed weevil and a detection method thereof, and relates to the technical field of biology. The PCR detection primer pair disclosed by the invention is high in specificity and sensitivity, can quickly and accurately identify whether the medicago sativa L. seeds contain the medicago sativa L. seeds or not, and can distinguish the medicago sativa L. seeds from other common pests such as medicago sativa L. beetles, medicago sativa L. aphids, medicago sativa L. prodenia litura, medicago sativa L. thrips and medicago sativa L. mucilage. The PCR primer combination disclosed by the invention can be used for simultaneously detecting a large batch of medicago sativa seeds, is simple and convenient to operate, and can be used for completing detection in a relatively short time under a laboratory condition; besides, the detection method is suitable for various alfalfa varieties, is wide in application range, is beneficial for alfalfa seed inspection personnel to inspect whether the alfalfa seeds are mixed with the alfalfa seed or not, can reduce the labor and time cost, and has a good application prospect.

Description

Technical Field

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

[0002] Alfalfa (Medicago sativa L.) is known as the "king of forage grass" in the world for its high quality, high yield, high feeding value (crude protein content 21%), good palatability, and wide range of uses. As a high-value crop, it can also be used to restore pastures due to its well-developed root system and ability to reduce soil erosion. Therefore, alfalfa has important economic and ecological value.

[0003] With the increase in the cultivated area, alfalfa pests are becoming increasingly serious, resulting in a significant decline in alfalfa production worldwide. These pests include alfalfa seed weevils, alfalfa weevils, alfalfa aphids, alfalfa cutworms, alfalfa thrips, alfalfa armyworms, alfalfa leafminers, alfalfa yellow flea beetles, etc. They directly cause the growth of alfalfa to be stunted and the yield to decrease by feeding on the leaves, stems and roots of the plants. In addition, the feeding of pests not only reduces the photosynthesis of plants, but also may cause the nutritional quality of plants to decrease. The presence of pests also increases the risk of plant infection. For example, some pests are transmitters of pathogens, which will spread pathogens such as viruses to healthy plants, causing greater damage. In addition, the reproduction and development of pests often cause ecological imbalance, affect biodiversity, and may lead to the invasion of new pest populations.

[0004] The clover weevil (Tychius medicaginis Bris.) belongs to the order Coleoptera, family Elephantiaceae. Figure 1 As shown in the figure. The larvae of the alfalfa seed weevil will eat the seeds, while the adults will gnaw on the mesophyll, damage the flower buds, calyx and corolla, and feed on the inner part of the floral organs. When the pods are formed, the female insects lay eggs in the young pods. After the larvae hatch, they bore into the seeds and excrete black feces to fill the pods, seriously damaging the alfalfa yield and seed quality. In severely damaged alfalfa fields, the seed yield may drop by more than 50%, which not only reduces farmers' income, but also increases the impact on the feed industry. With the rapid development of my country's animal husbandry, relevant ports import multiple batches of alfalfa hay and alfalfa seeds every year. Once the pest is introduced, the harm is very serious, and quarantine is the most economical and effective way to prevent the pest from colonizing and spreading. Accurate, rapid and effective detection methods are the technical basis for achieving effective quarantine, but there is no accurate, rapid and effective detection method for alfalfa seed weevil at present.

[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 alfalfa seed weevils and a detection method thereof. The kit and the detection method of the present invention can detect alfalfa seed weevils quickly, efficiently and accurately.

[0007] In a first aspect, the present invention provides a PCR primer pair for detecting alfalfa seed weevil, wherein the sequence of the forward primer of the PCR primer pair is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO.2.

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

[0009] In a third aspect, the present invention provides a kit for detecting alfalfa seed weevil, comprising the above-mentioned PCR primer pair.

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

[0011] In a fifth aspect, the present invention provides a method for detecting whether alfalfa seeds contain alfalfa seed weevils, comprising:

[0012] Extract genomic DNA from alfalfa seeds, then use the extracted DNA as a template to perform PCR amplification using the above kit, and then perform agarose gel electrophoresis on the PCR product; if bands appear in the electrophoresis result, the alfalfa seeds contain the alfalfa seed weevil; if no bands appear, the alfalfa seeds do not contain the alfalfa seed weevil.

[0013] The present invention has the following beneficial effects:

[0014] (1) The PCR detection primer pair of the present invention has high specificity and sensitivity, and can quickly and accurately identify whether alfalfa seeds contain alfalfa weevils, and can distinguish alfalfa weevils from other common pests such as alfalfa weevils, alfalfa aphids, alfalfa armyworms, alfalfa thrips and alfalfa armyworms;

[0015] (2) The detection of alfalfa seed weevils by the PCR primer combination of the present invention can simultaneously detect a large number of alfalfa seeds, and the operation is simple and the detection can be completed in a short time under laboratory conditions;

[0016] (3) The detection method provided by the present invention is applicable to a variety of alfalfa varieties and has a wide range of applications. At the same time, the detection method of the present invention helps alfalfa seed inspectors to inspect whether alfalfa seeds are mixed with alfalfa seed weevils, which can reduce labor and time costs and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 This is the information about the alfalfa seed weevil. Figure A shows the damage of the alfalfa seed weevil to alfalfa seeds; Figure B is a real photo of the alfalfa seed weevil.

[0019] Figure 2 The PCR identification results of 14 alfalfa varieties and 5 common alfalfa pests for the presence of the alfalfa weevil;

[0020] Figure 3 The PCR identification results of the sensitivity of the alfalfa seed kit for different numbers of seeds of Gannong 4, Gannong 7, Jinneng 5010 and Zhonglan 1;

[0021] Figure 4 for Figure 3 Sequencing and comparison results of PCR products from 40 seeds of Zhonggannong No. 4 and Jinneng 5010. DETAILED DESCRIPTION

[0022] 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.

[0023] The alfalfa seed weevil belongs to the family of elephants, and is named after the damage its larvae do to alfalfa and sweet clover seeds. The larvae of the alfalfa seed weevil eat the seeds, and the female lays eggs in young pods. After the larvae hatch, they bore into the seeds and excrete black feces that fill the pods, thus harming alfalfa yield and seed quality. Figure 1 As shown, 1A shows the destructiveness of alfalfa seed weevils to alfalfa seeds, and 1B is a real photo of the alfalfa seed weevils.

[0024] The present invention designs a PCR detection primer pair for alfalfa seed weevil, and the primer pair is:

[0025] TM-F (5′-3′): AAATTTATTTTATTTTTGGGTCTTGAG-SEQ ID NO.1;

[0026] TM-R (5′-3′): TTACAATCCTACTTATTAATAAAAGAGAAATTC-SEQ ID NO. 2.

[0027] Based on the above primer pair, the present invention can provide a detection kit for alfalfa seed weevil, which comprises the above primer pair and other conventional reagents for PCR amplification, including PCR amplification reagents, DNA templates and ddH2O.

[0028] Among them, the PCR amplification reagent includes Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, and other commonly used auxiliary reagents.

[0029] 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.

[0030] By amplifying the DNA of alfalfa seeds with or without alfalfa seed weevils, it was found that the target band could be amplified in the test sample only when alfalfa seed weevils were present.

[0031] Since the above detection primers or kits can be used to identify whether alfalfa seeds contain alfalfa weevils, the present invention also provides a method for detecting whether alfalfa seeds contain alfalfa weevils, which comprises:

[0032] (1) Extracting genomic DNA from alfalfa seeds to be tested;

[0033] (2) using the DNA obtained in step (1) as a template and the above primer pair to perform a PCR amplification reaction;

[0034] (3) taking the PCR amplification product and detecting it by agarose gel electrophoresis;

[0035] (4) Judging whether the alfalfa seeds to be tested contain alfalfa seed weevils based on whether characteristic bands appear in the electrophoresis bands: if bands appear in the electrophoresis results, the alfalfa seeds contain alfalfa seed weevils; if no bands appear, the alfalfa seeds do not contain alfalfa seed weevils.

[0036] In the above detection method, the reaction system of PCR amplification is: 12.5 μL of PCR amplification reagent, 1.0 μL of forward primer, 1.0 μL of reverse primer, 0.5 μL of template DNA, and 10 μL of ddH2O; wherein the concentration of the DNA template is <600 ng / μL; and the concentrations of the forward primer and the reverse primer are 10 μM.

[0037] The reaction program of PCR amplification was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 54°C for 15 s, extension at 72°C for 10 s, 35 cycles, and extension at 72°C for 5 min.

[0038] Experiments have shown that the detection method provided by the present invention can quickly, efficiently and accurately identify whether alfalfa seeds are mixed with alfalfa seed weevils.

[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 weevils, and its components include: PCR amplification primer pair, 2×RapidTaq Master Mix (Vazyme Biotech Co., Ltd; Nanjing, China; item number:

[0042] P222-01), DNA template and ddH2O.

[0043] Among them, the PCR amplification primer pair is:

[0044] TM-F (5′-3′): AAATTTATTTTATTTTTGGGTCTTGAG-SEQ ID NO.1;

[0045] TM-R (5′-3′): TTACAATCCTACTTATTAATAAAAGAGAAATTC-SEQ ID NO. 2.

[0046] Example 2

[0047] This embodiment is a method for detecting whether alfalfa seeds contain alfalfa seed weevils, which is detected using the detection kit in Example 1, and the specific steps are as follows:

[0048] S1. Germinate alfalfa seeds and extract leaf genomic DNA for later use;

[0049] S2. Collect alfalfa seeds and extract genomic DNA for later use;

[0050] S3. Using the primers specifically for alfalfa seeds in Example 1, perform PCR detection on the genomic DNA of leaves and seeds respectively.

[0051] The total volume of the PCR detection system was 25 μL, including 12.5 μL of 2×Rapid Taq Master Mix, 0.5 μL of DNA template (concentration < 600 ng / μL), 1 μL of primer TM-F (concentration 10 nM), 1 μL of primer TM-R (concentration 10 nM), and 10 μL of ddH2O.

[0052] The reaction procedure of PCR detection is as follows: (1) pre-denaturation at 95°C for 3 min; (2) denaturation at 95°C for 15 s; (3) annealing at 54°C for 15 s; (4) extension at 72°C for 10 s; (5) (2) to (4) steps are cycled 35 times; (6) extension at 72°C for 5 min;

[0053] S4. Use 1.4% agarose gel for detection, weigh 1.4g agarose, add 100mL of prepared 50×TAE, heat in a microwave oven for about 3min until the agarose is completely dissolved, add 10μL of nucleic acid dye and shake well, then pour the agarose solution into the gel mold, solidify for 40min, run the gel at 130V in a vertical plate electrophoresis instrument for 25min, and then observe the results under a gel imager.

[0054] Experimental Example 1

[0055] This experiment is a specificity verification test of the kit in Example 1, and is as follows:

[0056] S1. Germinate different varieties of alfalfa seeds and extract leaf genomic DNA for later use;

[0057] S2. Collect different varieties of alfalfa seeds and extract genomic DNA for later use;

[0058] S3. Using the primers specific for alfalfa seed weevil in Example 1, perform PCR detection on the genomic DNA of seeds and leaves.

[0059] The total volume of the PCR detection system was 25 μL, including 2×Rapid Taq Master Mix 12.5 μL, DNA template (concentration < 600 ng / μL) 0.5 μL, primer TM-F (concentration 10 nM) 1 μL, primer TM-R (concentration 10 nM) 1 μL, and ddH2O 10 μL.

[0060] The amplification conditions were as follows: (1) pre-denaturation at 95°C for 3 min; (2) denaturation at 95°C for 15 s; annealing at 54°C for 15 s; (4) extension at 72°C for 10 s; (5) (2) to (4) steps were cycled 35 times; (6) extension at 72°C for 5 min;

[0061] S4. Use 1.4% agarose gel for detection, weigh 1.4g agarose, add 100mL of prepared 50×TAE, heat in a microwave oven for about 3min until the agarose is completely dissolved, add 10μL of nucleic acid dye and shake well, then pour the agarose solution into the gel mold, solidify for 40min, run the gel at 130V in a vertical plate electrophoresis instrument for 25min, and then observe the results under a gel imager.

[0062] The information of alfalfa varieties and five common alfalfa pests tested in this experiment are shown in Table 1:

[0063] Table 1 Information on 14 alfalfa varieties tested and 5 common alfalfa pests

[0064]

[0065] Figure 2 In Figures A and B, 1-14 represent genomic DNA from 20 seeds and leaves of different alfalfa varieties; "+" represents alfalfa seed weevil (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. Take 1, 10, 20, 40 and 60 alfalfa seeds of Gannong No. 4, Gannong No. 7, Jinneng 5010 and Zhonglan No. 1 respectively and re-extract genomic DNA for PCR detection.

[0070] The total volume of the PCR detection system was 25 μL, including 12.5 μL of 2× Rapid Taq Master Mix, 0.5 μL of DNA template, 1 μL of primer TM-F, 1 μL of primer TM-R, and 10 μL of ddH2O.

[0071] 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; the concentrations of primers TM-F and TM-R are both 10nM.

[0072] The amplification conditions were as follows: (1) pre-denaturation at 95°C for 3 min; (2) denaturation at 95°C for 15 s; annealing at 54°C for 15 s; (4) extension at 72°C for 10 s; (5) steps (2) to (4) were cycled 35 times; and (6) extension at 72°C for 5 min.

[0073] S2. Use 1.4% agarose gel for detection, weigh 1.4g agarose, add 100mL of prepared 50×TAE, heat in a microwave oven for about 3min until the agarose is completely dissolved, add 10μL of nucleic acid dye and shake well, then pour the agarose solution into the gel mold, solidify for 40min, run the gel at 130V in a vertical plate electrophoresis instrument for 25min, and then observe the results under a gel imager.

[0074] 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, Gannong 4, Gannong 7, Jinneng 5010 and Zhonglan 1; "+" represents alfalfa seed (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 of three biological replicates.

[0075] Figure 3 It can be seen from AB that the genomic DNA of 60, 40, 20, 10 and 1 seeds of Gannong No. 4 and Gannong No. 7 can detect bands; the genomic DNA of 60 and 40 seeds of Jinneng 5010 can detect bands, while the genomic DNA of 20, 10 and 1 seeds cannot detect bands; the genomic DNA of 60, 40, 20 and 10 seeds of Zhonglan No. 1 can detect bands. According to the above results, it can be concluded that even if one alfalfa seed is used as a sample, the detection primer of the present invention can detect whether some varieties contain alfalfa seed weevils, which also proves that the detection primer of the present invention has a high detection sensitivity.

[0076] 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.

[0077] 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 pair for detecting alfalfa seed weevil, characterized in that: The sequence of the forward primer of the PCR primer pair is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO.

2.

2. Use of the PCR primer pair as claimed in claim 1 in preparing a kit for detecting alfalfa seed weevil.

3. A kit for detecting alfalfa seed weevils, characterized in that: Comprising the PCR primer pair described in 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 weevils.

7. The application according to claim 6, characterized in that: The application includes identifying whether alfalfa seeds contain the alfalfa weevil.

8. A method for detecting whether alfalfa seeds contain alfalfa weevils, 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 bands appear in the electrophoresis results, the alfalfa seeds contain the alfalfa seed weevil; if no bands appear, the alfalfa seeds do not contain the alfalfa seed weevil.

9. The method according to claim 8, characterized in that The reaction system for PCR amplification was: 12.5 μL of PCR amplification reagent, 1.0 μL of forward primer, 1.0 μL of reverse primer, 0.5 μL of template DNA, and 10 μL of ddH2O; The concentration of the DNA template is less than 600 ng / μL; the concentration of the forward primer and the reverse primer is 10 μM.

10. The method according to claim 8, characterized in that The reaction program of PCR amplification was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 54°C for 15 s, extension at 72°C for 10 s, 35 cycles, and extension at 72°C for 5 min.

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