Kit for identifying the alfalfa weevil and method of detection thereof
By designing PCR detection primer pairs (TM-F and TM-R) and kits, and combining PCR amplification and agarose gel electrophoresis, a rapid, efficient, and accurate detection of alfalfa seed weevils was achieved. This solves the problem of insufficient detection methods in existing technologies, is applicable to a variety of alfalfa varieties, and reduces detection costs and time.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2026-03-27
AI Technical Summary
The lack of accurate, rapid, and effective detection methods for alfalfa seed weevils in current technology leads to a decline in alfalfa yield and seed quality, affecting farmers' income and the feed industry.
A PCR detection primer pair (TM-F and TM-R) and corresponding kit were designed. By extracting genomic DNA from alfalfa seeds for PCR amplification and using agarose gel electrophoresis to detect the presence of characteristic bands, a rapid, efficient, and accurate detection of alfalfa seed weevils can be achieved.
It can quickly and accurately identify whether alfalfa seeds contain alfalfa seed weevils, distinguishing them from other pests. It is applicable to a variety of alfalfa varieties, reduces labor and time costs, and has broad application prospects.
Smart Images
Figure CN119220707B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular, to a kit for detecting and identifying Tychius medicaginis and a detection method thereof. BACKGROUND
[0002] Medicago sativa L. has the characteristics of high quality, high yield, high feeding value (crude protein content 21%), good palatability, and wide application, and is known as the "king of forage grass" in the world. As a high-value crop, it also has the ability to reduce soil erosion due to its extensive root system, and can be used for pasture restoration. Therefore, Medicago sativa L. has important economic and ecological values.
[0003] With the increase of planting area, the pest of Medicago sativa L. is becoming more and more serious, which leads to a significant decrease in the yield of Medicago sativa L. worldwide. These pests include Tychius medicaginis, Phyllobius pyri L., Aphis medicaginis Buckton, Spodoptera exigua Hübner, Thrips tabaci Lindeman, Mythimna separata Walker, Agromyza oranae Rondani, and Phyllotreta striolata Fabricius, etc. They directly cause the growth of Medicago sativa L. to be hindered and the yield to be decreased 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 can lead to the decrease of the nutritional quality of plants. The presence of pests also increases the risk of plant infection. For example, some pests are the spreaders of pathogens, which can spread viruses and other pathogens to healthy plants, thereby 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] Tychius medicaginis Bris. belongs to Coleoptera and Curculionidae, and its morphology is shown in Figure 1 The larvae of Tychius medicaginis Bris. will bore into seeds, and the adults will eat leaf flesh, damage flower buds, sepals and corolla, and eat the inside of the flower organ when it is in the process of forming pods. The female insects lay eggs in the young pods, and the larvae hatch and bore into the seeds, and fill the pods with black feces, which seriously damages the yield and seed quality of Medicago sativa L. The yield of seeds in severely damaged Medicago sativa L. fields can be reduced by more than 50%, which not only reduces the income of farmers, but also increases the impact on the feed industry. With the rapid development of animal husbandry in China, multiple batches of Medicago sativa L. hay and Medicago sativa L. seeds are imported at relevant ports every year. Once this pest is introduced, it will cause very serious damage. Quarantine is the most economical and effective method to prevent the pest from settling and spreading. However, there is currently no accurate, rapid and effective detection method for Tychius medicaginis.
[0005] Therefore, the present application is proposed. SUMMARY
[0006] The purpose of the present application is to provide a kit for detecting alfalfa weevil and a detection method thereof, which can quickly, efficiently and accurately detect alfalfa weevil.
[0007] In a first aspect, the present application provides a PCR primer pair for detecting alfalfa weevil, wherein the sequence of the forward primer of the PCR primer pair is shown in SEQ ID NO. 1, and the sequence of the reverse primer is shown in SEQ ID NO. 2.
[0008] In a second aspect, the present application provides the use of the above-mentioned PCR primer pair in the preparation of a kit for detecting alfalfa weevil.
[0009] In a third aspect, the present application provides a kit for detecting alfalfa weevil, which comprises the above-mentioned PCR primer pair.
[0010] In a fourth aspect, the present application provides the use of the above-mentioned kit in the detection of alfalfa weevil.
[0011] In a fifth aspect, the present application provides a method for detecting whether alfalfa weevil is contained in alfalfa seeds, which comprises:
[0012] extracting the genomic DNA of alfalfa seeds, then using the extracted DNA as a template to perform PCR amplification by using the above-mentioned kit, and then performing agarose gel electrophoresis detection on the PCR product; if a band appears in the electrophoresis result, then the alfalfa seeds contain alfalfa weevil; if no band appears, then the alfalfa seeds do not contain alfalfa weevil.
[0013] The present application has the following beneficial effects:
[0014] (1) The PCR detection primer pair of the present application has high specificity and sensitivity, and can quickly and accurately identify whether alfalfa weevil is contained in alfalfa seed, and can distinguish alfalfa weevil from other common pests such as alfalfa weevils, alfalfa aphids, alfalfa looper moths, alfalfa thrips and alfalfa stick insects, etc.
[0015] (2) The detection of alfalfa weevil by using the PCR primer combination of the present application can simultaneously detect a large number of alfalfa seed, and the operation is simple, which can complete the detection in a short time under laboratory conditions;
[0016] (3) The detection method provided by the present application is suitable for a variety of alfalfa varieties, and has a wide application range, and at the same time, the detection method of the present application is helpful for alfalfa seed inspectors to detect whether alfalfa weevil is mixed in alfalfa seed, which can reduce the labor and time cost, and has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.
[0018] Figure 1 For the related information of alfalfa weevil, Fig. A is the damage of alfalfa weevil to alfalfa seeds; Fig. B is a real photo of alfalfa weevil;
[0019] Figure 2 PCR identification results of whether 14 alfalfa varieties and 5 common alfalfa pests contain alfalfa weevil;
[0020] Figure 3 PCR identification results of the reagent kit sensitivity of different numbers of seeds of Gannong No. 4, Gannong No. 7, Jingneng 5010 and Zhonglan No. 1;
[0021] Figure 4 Figure 3 Sequencing and alignment results of PCR products of 40 seeds in Gannong No. 4 and Jingneng 5010. DETAILED DESCRIPTION
[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased in the market.
[0023] Alfalfa weevil belongs to Curculionidae, and is named because the larvae damage alfalfa and sweet clover seeds. The alfalfa weevil larvae will eat the seeds, the female insects lay eggs in the young pods, the larvae hatch, burrow into the seeds, and fill the pods with black feces, thereby endangering the yield and quality of alfalfa. As shown in the drawings, Fig. 1A shows the damage of alfalfa weevil to alfalfa seeds, and Fig. 1B is a real photo of alfalfa weevil. Figure 1
[0024] The present application designs a pair of PCR detection primers for alfalfa weevil, and the pair of primers is as follows:
[0025] TM-F (5'-3'): AACATTATATTTTATTTTTGGGTCTTGAG-SEQ ID NO. 1;
[0026] TM-R (5'-3'): TTACAATCCTACTTATTAATAAAAGAGAAATTC- SEQ ID NO. 2.
[0027] Based on the above primer pair, the present application can provide a detection kit for alfalfa weevil, which contains the above primer pair and other conventional reagents for PCR amplification, including PCR amplification reagents, DNA template and ddH2O.
[0028] Among them, the PCR amplification reagents contain Taq DNA polymerase, dNTPs, MgCl2, reaction buffer and other commonly used auxiliary reagents.
[0029] In addition to using the PCR amplification reagents provided by the present application, the PCR reaction system can also select commercial products of the same kind, which can be used for PCR amplification by adding a specified amount of DNA template and primer before amplification. The prerequisite is that the positive control sample must be amplified to produce the correct band, and the negative control sample must not produce the amplified band.
[0030] Through amplification detection of alfalfa seed DNA with or without alfalfa weevil, it is found that only in the presence of alfalfa weevil, the detection sample can be amplified to produce the desired band.
[0031] Since the above detection primer or kit can identify whether the alfalfa seed contains alfalfa weevil, the present application also provides a method for detecting whether the alfalfa seed contains alfalfa weevil, which comprises:
[0032] (1) Extracting the genomic DNA of the alfalfa seed to be tested;
[0033] (2) Using the DNA obtained in step (1) as a template, using the above primer pair to perform PCR amplification reaction;
[0034] (3) Taking the PCR reaction amplification product and detecting it by agarose gel electrophoresis;
[0035] (4) According to whether the characteristic band appears in the electrophoresis band, judging whether the alfalfa seed to be tested contains alfalfa weevil: if the electrophoresis result appears a band, then the alfalfa seed contains alfalfa weevil; if no band appears, then the alfalfa seed does not contain alfalfa weevil.
[0036] In the above detection method, the reaction system of PCR amplification is: PCR amplification reagents 12.5 μL, forward primer 1.0 μL, reverse primer 1.0 μL, template DNA 0.5 μL, ddH2O 10 μL; wherein the concentration of DNA template is <600 ng / μL; the concentration of forward primer and reverse primer is 10 μM.
[0037] The reaction procedure of PCR amplification is as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 54℃ for 15 s, extension at 72℃ for 10 s, 35 cycles, and extension at 72℃ for 5 min.
[0038] Experiments prove that the detection method provided by the application can quickly, efficiently and accurately identify whether alfalfa seeds are mixed with Phyllotreta striolata.
[0039] The application will be further described below through specific examples, but it should be understood that these examples are only for more detailed description and should not be understood as limiting the application in any form.
[0040] Example 1
[0041] This example is a kit for detecting Phyllotreta striolata, and the components thereof include: a pair of PCR amplification primers, 2x RapidTaq Master Mix (Vazyme Biotech Co., Ltd; Nanjing, China; item number: P222-01), a DNA template and ddH2O.
[0042] P222-01), a DNA template and ddH2O.
[0043] The pair of PCR amplification primers are as follows:
[0044] TM-F (5'-3'): AACATTATATTTTATTTTTGGGTCTTGAG-SEQ ID NO. 1;
[0045] TM-R (5'-3'): TTACAATCCTACTTATTAATAAAAGAGAAATTC-SEQ ID NO. 2.
[0046] Example 2
[0047] This example is a method for detecting whether alfalfa seeds contain Phyllotreta striolata, which utilizes the detection kit in Example 1 for detection, and the specific steps are as follows:
[0048] S1. Germinate alfalfa seeds, and extract leaf genomic DNA for standby;
[0049] S2. Collect alfalfa seeds, and extract genomic DNA for standby;
[0050] S3. Utilize the alfalfa seed-specific primers in Example 1 to perform PCR detection on leaf and seed genomic DNA, respectively.
[0051] PCR detection system total volume is 25 μL, including 2 x Rapid Taq Master Mix 12.5 μL, DNA template (concentration < 600 ng / μL) 0.5 μL, primer TM-F (concentration of 10 nM) 1 μL, primer TM-R (concentration of 10 nM) 1 μL, ddH2O 10 μL.
[0052] PCR detection reaction procedure is: (1) 95℃ pre-denaturation 3min; (2) 95℃ denaturation 15s; (3) 54℃ annealing 15s; (4) 72℃ extension 10s; (5) (2)-(4) step cycle 35 times; (6) 72℃ extension 5min;
[0053] S4. Detection with 1.4% agarose gel, weigh 1.4g agarose, add 100mL prepared 50 x TAE, microwave oven heating 3min or so to agarose completely dissolved, add 10 μL nucleic acid dye, shake well, then pour the agarose solution into the gel mold, solidification 40min, in the vertical electrophoresis apparatus, 130V voltage run gel 25min, then observe the results under the gel imaging instrument.
[0054] Experimental example 1
[0055] The experiment is a specificity verification test of the kit in example 1, as follows:
[0056] S1. Germination of different varieties of alfalfa seeds, extraction of leaf genomic DNA for standby;
[0057] S2. Collect different varieties of alfalfa seeds, extract genomic DNA for standby;
[0058] S3. Use the alfalfa seed-specific primer in example 1 to detect the seed and leaf genomic DNA by PCR.
[0059] PCR detection system total volume is 25 μL, including 2 x Rapid Taq Master Mix 12.5 μL, DNA template (concentration < 600 ng / μL) 0.5 μL, primer TM-F (concentration of 10 nM) 1 μL, primer TM-R (concentration of 10 nM) 1 μL, ddH2O 10 μL.
[0060] Amplification conditions: (1) 95℃ pre-denaturation 3min; (2) 95℃ denaturation 15s; 54℃ annealing 15s; (4) 72℃ extension 10s; (5) (2)-(4) step cycle 35 times; (6) 72℃ extension 5min;
[0061] S4. Detection was performed with 1.4% agarose gel. 1.4 g agarose was weighed, 100 mL prepared 50xTAE was added, and the agarose was completely dissolved by heating in a microwave oven for about 3 min. 10 μL nucleic acid dye was added and shaken, and then the agarose solution was poured into a gel mold. The gel was solidified for 40 min, and then electrophoresis was performed in a vertical electrophoresis apparatus at a voltage of 130 V for 25 min. The results were observed under a gel imager.
[0062] The alfalfa varieties and 5 common alfalfa pest information detected in the experiment are shown in Table 1:
[0063] Table 1 14 alfalfa varieties and 5 common alfalfa pest information
[0064]
[0065] Figure 2 In the experiment, figures A and B, 1-14 represent the genomic DNA of different alfalfa varieties 20 seeds and leaves; wherein "+" represents alfalfa weevils (positive control); "-" is water (negative control); M is marker; 15-19 represent the genomic DNA of common alfalfa parasitic pests; 1-19 represent the names of alfalfa varieties and common alfalfa parasitic pests as shown in Table 1. All genomic DNA was extracted three times independently, and PCR determination was performed respectively. The gel picture is a random picture of one of the three biological repeats.
[0066] From Figure 2 it can be seen that 10 bands were detected in the seeds of 1-14 alfalfa varieties, and 4 bands were not detected; no band was detected in the DNA of leaves; no band was detected in common alfalfa pests.
[0067] Experimental Example 2
[0068] The experiment was a sensitivity test of the kit in Example 1, and the details were as follows:
[0069] S1. Another Gannong No. 4, Gannong No. 7, Jingneng 5010, Zhilian No. 1 alfalfa seeds were extracted according to the number of 1, 10, 20, 40, 60 respectively to extract genomic DNA for PCR detection.
[0070] The total volume of the PCR detection system was 25 μL, which included 2xRapid Taq Master Mix 12.5 μL, DNA template 0.5 μL, primer TM-F 1 μL, primer TM-R 1 μL, and ddH2O 10 μL.
[0071] The concentrations of the DNA templates were as follows: 1 seed at 153 ng / μL; 10 seeds at 191 ng / μL; 20 seeds at 305 ng / μL; 40 seeds at 387 ng / μL; and 60 seeds at 443 ng / μL. The concentrations of primers TM-F and TM-R were both 10 nM.
[0072] The amplification conditions were: (1) 95℃ pre-denaturation for 3 min; (2) 95℃ denaturation for 15 s; 54℃ annealing for 15 s; (4) 72℃ extension for 10 s; (5) (2) to (4) steps were repeated 35 times; (6) 72℃ extension for 5 min.
[0073] S2. For detection using 1.4% agarose gel, weigh 1.4g of agarose, add 100mL of prepared 50×TAE, microwave for about 3 minutes 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 40 minutes, run the gel in a vertical plate electrophoresis apparatus at 130V for 25 minutes, and then observe the results under a gel imaging system.
[0074] The results are as follows Figure 3 As shown in Figures A, B, C, and D, numbers 1-5 represent the PCR identification results of genomic DNA extracted from 60, 40, 20, 10, and 1 seeds of alfalfa varieties Gannong 4, Gannong 7, Jinneng 5010, and Zhonglan 1, respectively. "+" represents alfalfa seed weevils (positive control); "-" represents water (negative control); and M represents the marker. All genomic DNA samples were extracted three times independently and analyzed by PCR. The gel images are random images from one of the three biological replicates.
[0075] Figure 3 As shown in Figures AB, bands were detected in the genomic DNA of 60, 40, 20, 10, and 1 seeds of Gannong 4 and Gannong 7; bands were detected in the genomic DNA of 60 and 40 seeds of Jinneng 5010, but no bands were detected in the 20, 10, and 1 seeds; and bands were detected in the genomic DNA of 60, 40, 20, and 10 seeds of Zhonglan 1. Based on these results, it can be concluded that even using a single alfalfa seed as a sample, the detection primers of this invention can detect the presence of alfalfa seed pheromones in some varieties, demonstrating the high detection sensitivity of the detection primers of this invention.
[0076] right Figure 3 The PCR products that detected bands were sequenced and analyzed, and the results are as follows: Figure 4 As shown, the sequencing results have a 99% similarity to the template sequence.
[0077] The above merely provides the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the scope of the present application.
Claims
1. A PCR primer pair for detecting alfalfa seed weevils, 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. The detection object of the PCR primer pair comprises alfalfa seeds.
2. The PCR primer pair of claim 1 in the preparation of a kit for detecting alfalfa weevils.
3. A kit for detecting alfalfa weevils, comprising, The kit comprises the PCR primer pair of claim 1.
4. The kit of claim 3, wherein The kit further comprises PCR amplification reagents, a DNA template, and ddH2O.
5. The kit of claim 4, wherein The PCR amplification reagents comprise Taq DNA polymerase, dNTPs, MgCl2, and a reaction buffer.
6. The kit of any one of claims 3-5 in the detection of alfalfa weevils.
7. Use according to claim 6, characterized in that, The application comprises identifying whether alfalfa seeds contain alfalfa weevils.
8. A method for detecting whether alfalfa seeds contain alfalfa seed weevils, characterized in that, The kit comprises: Genomic DNA is extracted from alfalfa seeds, and then the extracted DNA is used as a template for PCR amplification using the kit of any one of claims 3-5, and then the PCR product is detected by agarose gel electrophoresis; If a band appears in the electrophoresis result, the alfalfa seeds contain alfalfa weevils; if no band appears, the alfalfa seeds do not contain alfalfa weevils.
9. The method of claim 8, wherein, The reaction system for PCR amplification is: PCR amplification reagents 12.5 μL, forward primer 1.0 μL, reverse primer 1.0 μL, template DNA 0.5 μL, and ddH2O 10 μL. The concentration of the forward primer and the reverse primer is 10 μM.
10. The method of claim 8, wherein, The reaction program for PCR amplification is: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 54℃ annealing for 15 s, 72℃ extension for 10 s, 35 cycles, and 72℃ extension for 5 min.