Primer set and method for LAMP detection of L1014F mutation site of sodium channel of pear borer

Abstract

The present invention relates to the field of molecular biotechnology, and specifically discloses a LAMP detection primer set and method for the L1014F mutation of the sodium ion channel of pear borer, wherein the LAMP detection primer set comprises a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP and a loop primer LooP‑B. The sodium ion channel of pear borer is amplified using the primer set, and the result can be determined after the amplification. The method for detecting the L1014F mutation site of the sodium ion channel of pear borer established using the detection primer set provided by the present invention has high sensitivity, a large detection range, good amplification effect, and accurate and reliable detection results. It is suitable for the rapid detection of pear borer, and is of great significance for the monitoring of the drug resistance of lepidopteran harmful insects and the early warning of drug resistance.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and particularly relates to a LAMP detection primer set for a sodium ion channel L1014F mutation site of a pear borer moth and a detection method established by using the primer set. Background Art

[0002] Grapholita molesta (Busck) belongs to the Tortricidae family of the Lepidoptera order. It is a serious worldwide fruit pest. Its larvae mainly eat the tender shoots and fruits of various Rosaceae fruit trees such as peach, pear, apple, and plum. It has 3-4 generations a year in the Northeast and other places, and 4-5 generations a year in the south. As the climate gradually warms, the number of generations shows an increasing trend. The first and second generation larvae eat peach shoots, and a small amount of damage to pear and apple shoots. As summer approaches, the rate of broken branches of fruit trees increases, which significantly affects the shape and vigor of the trees. As the fruits appear, the larvae begin to eat the tender parts of the fruits. The damage caused by the pear borer seriously affects the appearance and quality of the fruits, causing significant economic losses to the fruit industry.

[0003] At present, the control of pear borer mainly relies on chemical control, and the long-term and large-scale use of chemical agents has caused the development of pest resistance. At present, studies have shown that pear borer has developed resistance to pyrethroid pesticides, such as high-efficiency cyhalothrin. Target resistance produced by pyrethroid insecticides is called knockdown resistance, and target resistance is one of the important mechanisms for the development of insect resistance. Sodium ion channel genes are closely linked to knockdown resistance, and mutations in their genes are closely related to knockdown resistance to pyrethroid insecticides. Most of the mutations related to knockdown resistance occur in the S5 and S6 transmembrane helical fragments of the sodium ion channel domain II and the intracellular fragment joint of IIS4-IIIS1, and a small number occur in domains I, II and IV. Single amino acid mutations on IIS6 are the most common, among which L1014 mutations have the highest frequency, and mutate to F in houseflies and German cockroaches. In other species, L1014F may mutate to different amino acids, such as L1014S in Anopheles gambiae, L1014W / G in Aedes aegypti, and L1014H in housefly. Sodium channel mutations can cause the extension of sodium channel opening, extend the opening and closing of sodium channels, significantly prolong the transmembrane sodium ion flow, cause nerve excitability conduction disorders, and thus cause toxic effects, ultimately leading to the death of insects. Sodium channel mutations exist in different insects, but the detection of this mutation generally relies on PCR amplification sequencing analysis, but this detection generally takes a long time and requires gel electrophoresis and molecular sequencing, which is cumbersome and costly to operate, and is difficult to promote and apply in actual production. LAMP detection has the characteristics of strong specificity, high sensitivity, convenient operation, and rapid detection, and the result judgment is intuitive, easy to operate, does not rely on any special instruments and equipment, and only requires a constant temperature device, which is easy to promote and apply in production practice. In addition, the detection cost of the LAMP detection method is lower than that of traditional PCR technology, and it is more efficient than traditional PCR technology.

[0004] LAMP detection technology has been widely used in species identification and site mutation detection, such as Metarhizium anisopliae (CN115747369A), Trypanosoma evansi (CN101892313B), and Alyssa lucorum acetylcholinesterase (CN112609008A). However, LAMP detection of sodium channel mutations in Pear Borer has not been reported at home and abroad. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a LAMP detection primer set for the L1014F mutation site of the sodium ion channel of the pear borer. The detection method established using the primer set has high sensitivity, a large detection range, good amplification effect, and accurate and reliable detection results, and is suitable for the rapid detection of the pear borer.

[0006] The specific technical solutions provided by the present invention are as follows:

[0007] In a first aspect, the present invention provides a LAMP detection primer set for the sodium channel L1014F mutation site of the pear borer, the primer set comprising a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP and a loop primer LooP-B;

[0008] The nucleotide sequence of the forward outer primer F3 is shown in SEQ ID NO.1;

[0009] The nucleotide sequence of the reverse external primer B3 is shown in SEQ ID NO.2;

[0010] The nucleotide sequence of the forward inner primer FIP is shown in SEQ ID NO.3;

[0011] The nucleotide sequence of the reverse inner primer BIP is shown in SEQ ID NO.4;

[0012] The nucleotide sequence of the loop primer LooP-B is shown in SEQ ID NO.5.

[0013] In a second aspect, the present invention provides a kit comprising the above-mentioned LAMP detection primer set.

[0014] As a preferred embodiment of the present invention, the kit further comprises a DNA extraction reagent and necessary reagents for LAMP reaction.

[0015] The third aspect of the present invention provides use of the primer set or the kit in detecting the L1014F mutation of the sodium ion channel of the pear borer.

[0016] In a fourth aspect, the present invention provides a method for rapid detection of the L1014F mutation in the sodium channel of the pear borer, comprising the following steps:

[0017] Extraction of genomic DNA of pear borer;

[0018] A LAMP reaction system was established, and the genomic DNA was used as a template, and forward outer primer F3, reverse outer primer B3, forward inner primer FIP, reverse inner primer BIP and loop primer LooP-B were used as detection primers for amplification;

[0019] The amplification results were determined by agarose gel electrophoresis or fluorescent dye colorimetry.

[0020] As a preferred embodiment of the present invention, the process of determining the amplification result using the fluorescent dye colorimetric method is as follows: dye is added to the amplified product, and the color is observed on white paper. If the test tube appears sky blue, it indicates the presence of a sodium ion channel L1014F site mutation.

[0021] As a preferred embodiment of the present invention, the dye added to the fluorescent dye color development method is hydroxynaphthol blue HNB, and a color development reaction is performed. If the reaction system turns sky blue, it indicates that the sample to be tested contains the sodium ion channel L1014F mutation; if the reaction system is violet, it indicates that the sample to be tested does not contain the sodium ion channel L1014F mutation.

[0022] As a preferred embodiment of the present invention, the process of determining the amplification result by agarose gel electrophoresis is as follows: if a ladder-shaped band appears, it indicates that there is a mutation in the sodium ion channel site.

[0023] As a preferred embodiment of the present invention, the genomic DNA of the pear borer is extracted using a small amount of genomic DNA extraction kit of Beyotime.

[0024] As a preferred embodiment of the present invention, the LAMP amplification reaction system includes 1 μL of 8U / μL Bst DNA polymerase, 2.5 μL of 10×Bst DNA polymerase buffer, 2.5 μL of 10 mM dNTPs, and 5 μL of 5 mM betaine.

[0025] As a preferred embodiment of the present invention, in the LAMP amplification reaction system, the outer primers F3 and B3 are 10 μM each, the inner primers FIP and BIP are 40 μM each, the inner and outer primers are mixed and added to a total of 7 μL, the volume ratio of the outer primers F3, B3 to the inner primers FIP, BIP is 8:1, and the volume ratio of the outer primer F3 to the outer primer B3, and the inner primer FIP to the inner primer BIP is 1:1.

[0026] As a preferred embodiment of the present invention, the reaction conditions of the LAMP amplification reaction system are: 65° C. for 50 min.

[0027] Compared with the prior art, the present invention has the following significant advantages:

[0028] 1. The rapid molecular detection method established by using the detection primer set for the sodium channel L1014F mutation of the pear borer provided by the present invention has high sensitivity, simple operation, no need for nucleic acid extraction and sequencing analysis, short detection time, and can obtain the detection result within 1 to 2 hours, and can detect 10 -3 The template concentration sample can be directly observed, and the test results can be used to detect the mutation of the sodium ion channel of pear borer.

[0029] 2. The LAMP detection primers and detection kit for the sodium ion channel site mutation of the pear fruit borer provided by the present invention have high sensitivity, a large detection range, good amplification effect, and accurate and reliable detection results, and are suitable for the rapid detection of the sodium ion channel site mutation of the pear fruit borer.

[0030] 3. Pear borer is an important fruit pest. When it is seriously harmful, it will have a huge impact on the quality and quality of the fruit, resulting in huge yield and economic losses. The mutation of the sodium ion channel is related to the resistance to pyrethroids, but the detection of its mutation site is generally time-consuming, cumbersome and costly, and it is difficult to promote and apply it in actual production. The present invention establishes a LAMP detection method and designs a detection kit, which can be used for the molecular detection of the mutation site of the sodium ion channel of the pear borer, providing a theoretical basis and technical support for whether it develops drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0032] Figure 1 The present invention is a gel image of the LAMP reaction detection of the pear borer DNA; wherein different lanes represent, respectively, CK: negative control; Na1: original concentration DNA; Na2: DNA diluted 10 times; Na3: DNA diluted 100 times; Na4: DNA diluted 1000 times; M: Marker2000.

[0033] Figure 2 This is a LAMP fluorescence reaction diagram of the pear borer in the present invention; wherein, CK: negative control; Na1: original concentration DNA; Na2: DNA diluted 10 times; Na3: DNA diluted 100 times; Na4: DNA diluted 1000 times.

[0034] Figure 3 This is a gel image of the LAMP reaction detection of the field pear borer DNA in the present invention; wherein different lanes represent, respectively, CK: negative control; 1: DNA of field pear borer sample 1; 2: DNA of field pear borer sample 2; 3: DNA of field pear borer sample 1; 4: DNA of field pear borer sample 4; 5: DNA of field pear borer sample 5; M: Marker2000.

[0035] Figure 4 It is a LAMP fluorescence reaction diagram of field pear borer in the present invention; wherein, CK: negative control; 1: DNA of field pear borer sample 1; 2: DNA of field pear borer sample 2; 3: DNA of field pear borer sample 1; 4: DNA of field pear borer sample 4; 5: DNA of field pear borer sample 5.

[0036] Figure 5This is a gel image of the LAMP reaction detection of the pear borer DNA in the present invention; wherein different lanes represent, respectively, CK: negative control; 1-2: wild-type individuals; 3-5: mutant individuals; M: Marker2000.

[0037] Figure 6 This is a LAMP fluorescence reaction diagram of the pear borer in the present invention; wherein, CK: negative control; 1-2: wild-type individuals; 3-5: mutant individuals. DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Under the protection scope of the spirit and essence of the present invention, any modification or replacement of the method, steps or conditions of the present invention belongs to the scope of the present invention. It should be known that all other embodiments obtained by ordinary technicians in this field without creative work based on the embodiments of the present invention also belong to the scope of protection of the present invention.

[0039] Unless otherwise specified, the materials, reagents and methods used in the embodiments of the present invention are all conventionally operated and commercially available.

[0040] Example 1

[0041] 1. Design of LAMP primer combination for sodium channel site mutation

[0042] The gene sequence of the sodium channel of the pear borer Grapholita molesta (SEQ ID NO.6) was amplified and determined, and compared with the gene sequences of other insect species in the GenBank database. The LAMP primers were designed using the online LAMP primer design software Primer Explorer V5 software (http: / / primerexplorer.jp / lampv5e). The length of the target sequence amplified by LAMP should be controlled below 300 bp. The LAMP primers include the forward outer primer F3 (SEQ ID NO.1), the reverse outer primer B3 (SEQ ID NO.2), the forward inner primer FIP (SEQ ID NO.3), the reverse inner primer BIP (SEQ ID NO.4), and the loop primer LooP-B (SEQ ID NO.5). The primer design rules are the same as those of PCR. The inner primer length is usually more than 40 bases, and the outer primer is about 20 bases. In addition to the length and base composition of the primers, the loop structure of the primer and the target sequence needs to be considered. The specific sequences of each primer are as follows:

[0043] SEQ ID NO.1: 5'-ATTACGTGGACCGGTTTCC-3';

[0044] SEQ ID NO.2: 5'-ACAGGGCCAAGAAGAGGTTA-3';

[0045] SEQ ID NO.3:

[0046] 5'-TTCTCCCGCAGAGCACTCGGAAGGTGGAACTTCACCGACTTC-3';

[0047] SEQ ID NO.4:

[0048] 5'-GTGGGATTGCATGCTTGTCGGTTACCACAAAATTGCCAATGA-3';

[0049] SEQ ID NO.5: 5'-CTCCTGTATTCCCTTTTTCCTGG-3';

[0050] SEQ ID NO.6:

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] The bold part is the mutation site, that is, the sodium channel L1014F mutation of the pear borer is the mutation from C to T at the bold part of the above sequence;

[0057] Example 2

[0058] This example provides a rapid molecular detection method for the sodium channel L1014F of the pear borer. 1. Extraction of genomic DNA of the pear borer

[0059] 1. Prepare 1 pear borer larvae and place them in a 1.5 mL sterile enzyme-free tube. Add 50 μL of sample lysis solution A for grinding (grinding rod). Use 150 μL of sample lysis solution A to rinse the grinding rod.

[0060] 2. Add 20 μL proteinase K and incubate in 55°C water bath for 60 min;

[0061] 3. Add 4 μL 100 mg / mL RNase A, mix well and place at room temperature for 2 minutes;

[0062] 4. Shake vigorously for 30 seconds, add 200 μL sample lysis buffer B, shake and mix. Incubate in 70℃ water bath for 10 minutes;

[0063] 5. Add 200 μL of anhydrous ethanol and vortex for 15 seconds;

[0064] 6. Add all the liquid to a 1.5 mL centrifuge column and centrifuge at maximum speed for 1 min until all the liquid is centrifuged;

[0065] 7. Place the centrifuge column in a new 2 mL centrifuge tube, add 500 μL of washing solution I (add 7 mL of anhydrous propanol before use), centrifuge at maximum speed for 30 seconds, and discard the waste liquid;

[0066] 8. Place the centrifuge column in a new 2 mL centrifuge tube, add 600 μL of washing solution II (add 24 mL of anhydrous propanol before use), centrifuge at maximum speed for 30 seconds, and discard the waste liquid;

[0067] 9. Idle at maximum speed for 2 minutes;

[0068] 10. Place the centrifuge column in a new 1.5 mL centrifuge tube, add 50-200 μL of Elution Buffer preheated to 70°C, let stand at room temperature for 2 minutes, and centrifuge at maximum speed for 1 minute.

[0069] 11. Repeat step 10 to increase the DNA concentration, measure the concentration at last and store at -20℃.

[0070] 2. Establishment of LAMP detection reaction system

[0071] The DNA extracted in the above step was used as a template, and the primers in Example 1 were used as detection primers to establish a LAMP detection system for the sodium channel site mutation of the pear borer. The specific steps are as follows:

[0072] (1) Preparation of primer set mixture: dilute outer primers F3 and B3 to 10 μM, inner primers FIP and BIP to 40 μM, and loop primer LooP-B to 10 μM. Mix the inner and outer primers and add a total of 7 μL. The volume ratio of outer primers (F3, B3) to inner primers (FIP, BIP) is 8:1, the volume ratio of outer primer F3 to outer primer B3 is 1:1, the volume ratio of inner primer FIP to inner primer BIP is 1:1, and the volume ratio of outer primers (F3, B3), inner primers (FIP, BIP) and loop primer is 8:1:1.

[0073] (2) Establishment of LAMP reaction system: 2.5 μL of 10×LAMP Master Mix reaction mixture, 1 μL of 8UBst polymerase, 1 μL of DNA template (different dilution multiples), 5 μL of 5 mM betaine, and add sterile ultrapure water to make up to 25 μL. Before amplification, 2 μL of 250 μM HNB was added to the test tube, and finally 25 μL of paraffin oil was added to seal (to prevent false positives); the composition and content of substances in 2.5 μL of LAMP Master Mix reaction mixture were: dNTPs 10 mmol, pH 8.8 Tris-HCl 20 mmol, KCl 10 mmol, MgSO4 2 mmol, (NH4)2SO4 10 mmol, 0.1% TritonX-100.

[0074] (3) The above reaction system was placed in a water bath at 65°C for 50 min. After the amplification, 5 μL of the product was detected by 1.0% agarose gel electrophoresis. The electrophoresis detection conditions were: voltage 80 V, current 300 A, and detection time 50 min.

[0075] (4) Determination of amplification results: (a) Agarose gel electrophoresis shows ladder-shaped bands, indicating the presence of a mutation in the sodium ion channel site, see Figure 1 (b) Fluorescent dye colorimetric method: After amplification, place the product test tube on white paper for color observation. If the test tube appears sky blue, it indicates that there is a mutation at the sodium ion channel site. Figure 2 .

[0076] Example 3

[0077] In this example, samples of pear borer were collected from the field for rapid molecular detection of the sodium ion channel L1014F.

[0078] 1. Extraction of genomic DNA of Pear Borer in the Field

[0079] The genomic DNA of No. 1 to 5 single-headed pear borer adults collected in the field was extracted according to the genomic DNA extraction method described in Example 2.

[0080] 2. LAMP detection reaction

[0081] According to the LAMP reaction system established in Example 2, the reaction was carried out using the genomic DNA of the adult pear borer in step 1 as a template. The primer ratio, reaction conditions and gel electrophoresis conditions were also as described in step 2 of Example 2.

[0082] Figure 3 and Figure 4After LAMP isothermal amplification, the gel electrophoresis results of field pear borer samples 1, 2 and 4 showed ladder-shaped bands. At the same time, after LAMP amplification, the product test tubes showed a sky blue change on the white paper, indicating that there were sodium ion channel site mutations in field pear borer samples 1, 2 and 4. The LAMP reaction system and conditions in Example 2 can be used for the rapid detection of the L1014F site mutation of the sodium ion channel of field pear borer.

[0083] Comparative Example 1

[0084] Replacement of LAMP primers for detection

[0085] 1. Design of LAMP primer combination

[0086] According to the LAMP primer design principle, the primers used in Comparative Example 1 were designed using the LAMP primer design software in Example 1. The primers and the LAMP primers used in the present invention were designed based on the same sequence fragment.

[0087] The specific primer sequences are as follows:

[0088] F3-1: 5'-GTTTCCGGACGGAGACC-3', SEQ ID NO.7;

[0089] B3-1: 5'-ACAGGGCCAAGAAGAGGT-3', SEQ ID NO.8;

[0090] FIP-1:

[0091] 5'-CTCCGCAGAGCACTCGGAACGGTGGAACTTCACCGACTTC-3', SEQ ID NO.9;

[0092] BIP-1:

[0093] 5'-TGTGGGATTGCATGCTTGTCGGTACCACAAAATTGCCAATGA-3', SEQ ID NO.10;

[0094] Loop-B1: 5'-TGACGTCTCCTGTATTCCCTTTT-3', SEQ ID NO. 11.

[0095] 2. Extraction of genomic DNA from single-headed pear borer adult

[0096] The genomic DNA of single-headed pear borer adult moths No. 1 to 5 was extracted according to the genomic DNA extraction method described in Example 2.

[0097] 3. LAMP reaction

[0098] LAMP reaction system: dilute the outer primers F3-1 and B3-1 to 10 μM, the inner primers FIP-1 and BIP-1 to 40 μM, and the loop primer LooP-B1 to 10 μM. Mix the inner and outer primers and add a total of 7 μL. The volume ratio of the outer primers F3-1, B3-1 to the inner primers FIP-1, BIP-1 is 8:1, the volume ratio of the outer primer F3-1 to the outer primer B3-1, the inner primer FIP-1 to the inner primer BIP-1 is 1:1, and the volume ratio of the outer primer to the inner primer and the loop primer is 8:1:1. Add 2.5 μL of 10×LAMP PCR isothermal amplification reaction mixture, 1 μL of 8U Bst polymerase, 1 μL of circular primer LooP-B, 1 μL of DNA template, 5 μL of 5mM betaine to the reaction tube, and add sterile ultrapure water to 25 μL. Add 2 μL of 250 μM HNB color developer to the tube before amplification, and finally add 25 μL of paraffin oil to seal, and water bath at 65°C for 50 minutes. After the amplification, take 5 μL of the product and detect it by 1.0% agarose gel electrophoresis. The electrophoresis detection conditions are: voltage 80V, current 300A, and detection time 50 minutes.

[0099] like Figure 5 and 6 The test results showed that the pear borer individuals No. 1 to 2 were wild type, and the color of tubes No. 1 to 2 did not change. The pear borer individuals No. 3 to 5 contained the L1014F site mutation, but the reaction tubes No. 3 to 5 did not change color and remained purple, indicating that the normal LAMP reaction could not be carried out after the LAMP primer sequence was replaced.

[0100] The change of LAMP detection primers (inner primers, outer primers and loop primers) will not start the LAMP reaction. In the present invention, primer design is a limiting factor. Inappropriate primer sequences will lead to failure to perform correct molecular detection, so the primers provided by the present invention are specific.

[0101] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0102] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A LAMP detection primer set for the sodium channel L1014F mutation of pear borer, characterized in that: The primer set includes a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP and a loop primer LooP-B; The nucleotide sequence of the forward outer primer F3 is shown in SEQ ID NO.1; The nucleotide sequence of the reverse external primer B3 is shown in SEQ ID NO.2; The nucleotide sequence of the forward inner primer FIP is shown in SEQ ID NO.3; The nucleotide sequence of the reverse inner primer BIP is shown in SEQ ID NO.4; The nucleotide sequence of the loop primer LooP-B is shown in SEQ ID NO.

5.

2. A kit comprising the primer set according to claim 1.

3. The kit according to claim 2, characterized in that The kit includes a DNA extraction reagent and necessary reagents for LAMP reaction.

4. Use of the primer set according to claim 1 or the kit according to claim 2 in detecting the L1014F mutation of the sodium ion channel of the pear borer.

5. A method for detecting the L1014F mutation of the sodium ion channel of the pear borer using the primer set of claim 1, characterized in that: The steps include: Extraction of genomic DNA of pear borer; A LAMP reaction system was established, with the extracted genomic DNA as the template, and the forward outer primer F3, the reverse outer primer B3, the forward inner primer FIP, the reverse inner primer BIP and the loop primer LooP-B as the detection primers for amplification; The amplification results were determined by agarose gel electrophoresis or fluorescent dye colorimetry.

6. The method for detecting the L1014F mutation of the sodium ion channel of the pear borer according to claim 5, characterized in that: The process of using the fluorescent dye colorimetric method to determine the amplification results is as follows: adding dye to the amplified product and observing the color change. If sky blue appears, it indicates that there is a sodium ion channel L1014F site mutation in the sample to be tested; if violet appears, it indicates that there is no sodium ion channel L1014F mutation in the sample to be tested.

7. The method for detecting the L1014F mutation of the sodium ion channel of the pear borer according to claim 5, characterized in that: The process of determining the amplification results by agarose gel electrophoresis is as follows: if a ladder-shaped band appears, it indicates the presence of a sodium ion channel site mutation.

8. The method for detecting the L1014F mutation of the sodium ion channel of the pear borer according to claim 5, characterized in that: The LAMP amplification reaction system includes 1 μL of 8U / μL Bst DNA polymerase, 2.5 μL of 10×Bst DNA polymerase buffer, 2.5 μL of 10 mM dNTPs, and 5 μL of 5 mM betaine.

9. The method for detecting the L1014F mutation of the sodium channel of the pear borer according to claim 5, characterized in that: The mass concentrations of the outer primers F3 and B3 are both 10 μM, the mass concentrations of the inner primers FIP and BIP are both 40 μM, the total volume of the inner and outer primers is 7 μL, the volume ratio of the outer primers F3, B3 to the inner primers FIP, BIP is 8:1, and the volume ratio of the outer primer F3 to the outer primer B3, and the inner primer FIP to the inner primer BIP is 1:

1.

10. The method for detecting the L1014F mutation of the sodium ion channel of the pear borer according to claim 5, characterized in that: The reaction conditions of LAMP amplification reaction were: 65°C for 50 min.

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