LAMP Detection Primers and Method for Mutations in the GABA Receptor of the Oriental Fruit Moth

Abstract

The present invention belongs to the field of biotechnology and mainly relates to a LAMP detection primer and method for the mutation of the GABA receptor of the codling moth. The primers include a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP, a forward loop primer Loop-F, and a reverse loop primer Loop-B. Using the detection primers provided by the present invention, a rapid detection method for the A302S mutation of the GABA receptor of the codling moth can be established, and further determine whether the codling moth is resistant to abamectin. The LAMP detection primer set can be used for the detection of the mutation of the GABA receptor of the codling moth, with high sensitivity, a large detection range, and accurate and reliable results; the detection method is simple to operate, does not require nucleic acid extraction of the oriental fruit moth, has a short detection time, and the detection results are visualizable. The present invention can provide technical support for the resistance monitoring and early warning of the codling moth in the field, and also provide theoretical support for the integrated control of the oriental fruit moth.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and mainly relates to a LAMP detection primer and method for GABA receptor mutation of fruit borer. 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 branch breakage 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 and environmental pollution. Studies have shown that pear borer has developed resistance to a variety of pesticides such as pyrethroids, cyclopentadiene, o-formylaminobenzamides and antibiotics, such as highly effective chlorfenapyr and avermectin. Target site incompatibility is one of the important mechanisms for insect resistance, and mutations in GABA (γ-aminobutyric acid) receptors have also caused resistance in a variety of insects. GABA receptors are distributed throughout the nervous system and are ligand-gated ion channel receptors. The receptors have GABA recognition sites and specific binding sites for different ligands and compounds. The GABA receptors of insects are mainly present in the central nervous system and neuromuscular junctions, and include three subunits, RDL (resistance to dieldrin), GRD (GABA and glycine-like receptor of Drosophila), and LCCH3 (ligand-gated chloride channel homologue 3). The research on insect GABA receptors mainly focuses on the RDL subunit. The 302nd alanine mutation in the second transmembrane region of the RDL subunit is converted to serine (A302S), which leads to the generation of drug resistance.

[0004] At present, many mutations of GABA receptors have been found in insects, such as A302S, A302G, T350M and T350L mutations in Drosophila melanogaster; A302S and R300Q mutations in brown planthopper; A296S and V327I mutations in Anopheles gambiae; A302S in German cockroach; D472N in diamondback moth, etc. GABA receptor mutations exist in different insects, but the detection of this mutation generally relies on PCE amplification sequencing detection, but this detection generally takes a long time, the operation is cumbersome and the cost is high, and it is difficult to promote in actual production. LAMP detection has the characteristics of strong specificity, high sensitivity, convenient operation, and rapid detection. The result judgment is intuitive, the operation is simple, and it does not rely on any special instruments and equipment. Only a constant temperature device is required, 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 the traditional PCR technology, and it is more efficient than the traditional PCR technology.

[0005] LAMP detection technology has been widely used in species identification and site mutation detection, such as Ralstonia solanacearum (CN106893764A), Fusarium pyrifolia (CN106893763A), aphid sodium ion channel (CN116497099B) and green stink bug acetylcholinesterase (CN112609008A), etc. However, LAMP detection of GABA receptor mutations in fruit borer has not been reported at home and abroad. Summary of the invention

[0006] The purpose of the present invention is to provide a LAMP detection method for GABA receptor mutation of fruit borer and its application, which are used to provide intuitive judgment on the generation of drug resistance of fruit borer.

[0007] In view of the above objectives and technical problems, the present invention uses pear borer as the detection species and designs LAMP detection primers that can be used for LAMP detection of GABA receptor mutations of the borer.

[0008] The technical solution of the present invention is as follows:

[0009] In a first aspect of the present invention, a LAMP primer for detecting a GABA receptor mutation of a fruit borer is provided, wherein the primer set comprises a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP, a forward loop primer Loop-F and a reverse loop primer Loop-B, and the primer sequences are as follows:

[0010] F3: 5'-CGTTCTGGCTGAACCGAA-3';

[0011] B3: 5'-TTGGCCATATATCCTACTGTAGC-3';

[0012] FIP: 5'-GGTGGAGGACATGAGCGTTGTACGCCGGCGCGCGTGTCGC-3';

[0013] BIP: 5'-GCGCTGCCTAAGATCTCCTACGAGACCATGACGAAACAGGTG-3';

[0014] Loop-F: 5'-TGAGCACGGTGGTGACG-3';

[0015] Loop-B: 5'-TCAAGTCCATCGACGTCTACC-3'.

[0016] In a second aspect, the present invention provides a LAMP detection kit for GABA receptor mutation of fruit borer, comprising the primer set.

[0017] In a third aspect, the present invention provides a use of the primer set or the kit in detecting GABA receptor mutations in fruit borer.

[0018] Furthermore, the primer set or the detection kit is used to detect pear borer.

[0019] In a fourth aspect, the present invention provides a method for detecting a GABA receptor mutation of a pear borer, comprising the following steps:

[0020] Extracting genomic DNA from the sample to be tested;

[0021] Using the extracted DNA as a template, amplification is performed in a LAMP reaction system using the primer set;

[0022] Determine the amplification results.

[0023] Furthermore, the LAMP reaction system includes: 10 μM of outer primers F3 and B3, 40 μM of inner primers FIP and BIP, 10 μM of loop primers Loop-F and Loop-B, 2.5 μL of 10×LAMP reaction mixture, 1 μL of 8U Bst polymerase, 1 μL of DNA template, 5 μL of 5mM betaine, and ddH 2 O to 25 μL, then add 25 μL paraffin oil to seal;

[0024] Among them, the total amount of external primers F3, B3 and internal primers FIP, BIP added is 7 μL, the total amount of loop primers Loop-F and Loop-B added is 1 μL, and the ratio of the total volume of external primers F3, B3 to the total volume of internal primers FIP, BIP is 8:1, the volume ratios of external primer F3 and external primer B3, internal primer FIP and internal primer BIP, and loop primers Loop-F and Loop-B are all 1:1.

[0025] Furthermore, the composition and content of the substances in the 2.5 μL LAMP reaction system were: dNTPs 10 mmol, pH 8.8 Tris-HCl 20 mmol, KCl 10 mmol, MgSO 4 2 mmol, (NH4) 2 SO 4 10 mmol, 0.1% Triton X-100.

[0026] Furthermore, the amplification condition of the LAMP reaction was incubated at 65°C for 50 min.

[0027] Further, the amplification results were determined using the following method:

[0028] Fluorescent dye colorimetric method: Add the dye hydroxynaphthol blue HNB to the amplified product for colorimetric reaction. If the reaction system turns sky blue, it indicates that the sample to be tested contains the GABA receptor mutation of the fruit borer; if the reaction system turns violet, it indicates that the sample to be tested does not contain the GABA receptor mutation of the fruit borer; or

[0029] Gel electrophoresis: If the amplified product shows a characteristic ladder-shaped amplification band on the 1% agarose gel, it indicates that the sample to be tested has a GABA receptor mutation of the fruit borer; if no characteristic ladder-shaped amplification band appears, it indicates that the sample to be tested does not have a GABA receptor mutation of the fruit borer.

[0030] Beneficial effects:

[0031] (1) The LAMP detection primers and detection kit for GABA receptor mutation of fruit borer provided by the present invention have high sensitivity, large detection range, good amplification effect, and accurate and reliable detection results.

[0032] (2) The method for detecting GABA receptor mutations in fruit borer using the LAMP detection method provided by the present invention has high sensitivity, simple operation, and short detection time. The detection results can be obtained within 1 to 2 hours, and 10 -4 The template concentration sample is 100%, and the detection results are visualized, which can be used to detect the GABA receptor mutation of the fruit borer. The results of fluorescent LAMP and chromogenic LAMP are consistent. Compared with the traditional PCR method, the detection efficiency is greatly improved, which is suitable for the rapid detection of pear fruit borer.

[0033] (3) The present invention establishes a LAMP detection method for the GABA receptor mutation of the fruit borer and designs a detection kit, which can be used for the molecular detection of the GABA receptor mutation of the fruit borer. The detection results provide a theoretical basis for whether the fruit borer in the field develops drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a gel image of the LAMP reaction of the DNA of the pear borer; CK: negative control; Ga1: original concentration DNA; Ga2: 10-fold diluted DNA; Ga3: 100-fold diluted DNA; Ga4: 1000-fold diluted DNA; Ga5: 10000-fold diluted DNA; M: Marker2000;

[0035] Figure 2 This is the LAMP fluorescence reaction diagram of pear borer; CK: negative control; Ga1: original concentration DNA; Ga2: 10-fold diluted DNA; Ga3: 100-fold diluted DNA; Ga4: 1000-fold diluted DNA; Ga5: 10,000-fold diluted DNA.

[0036] Figure 3 This is a gel image of the LAMP reaction of the DNA of the pear borer in the field; CK: negative control; G1: pear borer sample 1 in the field; G2: pear borer sample 2 in the field; G3: pear borer sample 3 in the field; G4: pear borer sample 4 in the field; G5: pear borer sample 5 in the field; M: Marker2000;

[0037] Figure 4 This is the LAMP fluorescence reaction diagram of pear borer in the field; CK: negative control; G1: G1: field pear borer sample 1; G2: field pear borer sample 2; G3: field pear borer sample 3; G4: field pear borer sample 4; G5: field pear borer sample 5.

[0038] Figure 5 This 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.

[0039] 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

[0040] In order to better understand the content of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0041] Unless otherwise specified, the materials, reagents and methods used in the embodiments of the present invention are all conventionally operated and commercially available.

[0042] Example 1

[0043] Design of LAMP primer set for GABA receptor mutation

[0044] The gene sequence of the GABA receptor of Grapholita molesta (Busck) was determined (SEQ ID NO: 7) and compared with the gene sequences of other species in the GenBank database. The online LAMP primer design software Primer Explorer V5 software ( http: / / primerexplorer.jp / lampv5e ) Design LAMP primers, the LAMP primers include a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP, a forward loop primer Loop-F and a reverse loop primer Loop-B, and the specific primer sequences are as follows:

[0045] F3: 5'-CGTTCTGGCTGAACCGAA-3', SEQ ID NO: 1;

[0046] B3: 5'-TTGGCCATATATCCTACTGTAGC-3', SEQ ID NO: 2;

[0047] FIP: 5'-GGTGGAGGACATGAGCGTTGTACGCCGGCGCGCGTGTCGC-3', SEQ ID NO: 3;

[0048] BIP: 5'-GCGCTGCCTAAGATCTCCTACGAGACCATGACGAAACAGGTG-3', SEQ IDNO: 4;

[0049] Loop-F: 5'-TGAGCACGGTGGTGACG-3', SEQ ID NO: 5;

[0050] Loop-B: 5'-TCAAGTCCATCGACGTCTACC-3', SEQ ID NO: 6.

[0051] SEQ ID NO: 7:

[0052]

[0053]

[0054] in,

[0055] The bold part of the sequence is the mutation site of the GABA receptor of the pear borer, which is the mutation of base G to T.

[0056] Example 2

[0057] Extraction of Genomic DNA from Pear Borer

[0058] The TIANamp Genomi DNA Kit was used to extract the genomic DNA of the pear borer. The specific steps are as follows:

[0059] 1. Prepare 1 adult of Pear Borer and place it in a 1.5 mL sterile tube. Add 50 μL of buffer GA for grinding (grinding rod). Use 150 μL of buffer GA to rinse the grinding rod.

[0060] 2. Add 4 μL RNase A (100 mg / ml) solution, shake for 15 seconds, and leave at room temperature for 5 minutes;

[0061] 3. Add 20 μL Proteinase K solution, shake and mix, and place in a 56°C water bath for 30 min;

[0062] 4. Add 200 μL of buffer GB, mix thoroughly by inversion, and place in a 70°C water bath for 10 min;

[0063] 5. Add 200 μL of anhydrous ethanol and oscillate to mix for 15 seconds (flocculate precipitation may occur);

[0064] 6. Transfer the solution and flocculent precipitate obtained in step 5 into adsorption column CB3, centrifuge at maximum speed for 1 min, and discard the waste liquid;

[0065] 7. Add 500 μL of buffer GD (with anhydrous ethanol added) to the adsorption column and centrifuge at maximum speed for 1 min, then discard the waste liquid;

[0066] 8. Add 600 μL of rinse solution PW (with anhydrous ethanol added) to the adsorption column and centrifuge at maximum speed for 1 min, then discard the waste liquid;

[0067] 9. Repeat step 10, and then idle at maximum speed for 2 minutes;

[0068] 10. Place the centrifuge column in a new 1.5 mL sterile centrifuge tube, add 50-100 μL of elution buffer, and let stand at room temperature for 2 minutes; centrifuge at maximum speed for 1 minute;

[0069] 11. Repeat step 12 to increase the DNA concentration, measure the concentration and store at -20°C.

[0070] Example 3

[0071] Establishment of LAMP detection reaction system

[0072] The DNA extracted in the above Example 2 was used as a template, and the primer set designed in Example 1 was used as a detection primer to establish a LAMP detection system for the GABA receptor site mutation of the pear borer. The reaction system included: the mass concentrations of the outer primers F3 and B3 were both 10 μM, the mass concentrations of the inner primers FIP and BIP were both 40 μM (7 μL was added after the inner and outer primers were mixed, the ratio of the total volume of the outer primers F3 and B3 to the total volume of the inner primers FIP and BIP was 8:1, the outer primer F3 and the outer primer B3 were added in equal amounts, and the inner primer FIP and the inner primer BIP were added in equal amounts), 1 μL of each 10 μM of the forward loop primer Loop-F and the reverse loop primer Loop-B was added, 2.5 μL of a 10×LAMP PCR isothermal amplification reaction mixture, 8 U 1μL of Bst polymerase, 1.0μL of DNA template (different dilution multiples), 5μL of 5mM betaine, add sterile ultrapure water to 25μL, add 1μL of 250μM HNB colorimetric agent to the test tube before amplification, and finally add 25μL of paraffin oil to seal (to prevent aerosol contamination and avoid false positives), and water bath at 65℃ for 60min. The composition and content of the substance in the 2.5μLLAMP PCR isothermal amplification reaction mixture are: dNTPs 10mmol, pH 8.8 Tris-HCl 20mmol, KCl 10mmol, MgSO 4 2 mmol, (NH4) 2 SO 4 10 mmol, 0.1% Triton X-100.

[0073] After the amplification, 5 μL of the product was taken and detected by 1.0% agarose gel electrophoresis. The electrophoresis detection conditions were: voltage 80 V, current 400 A, and detection time 50 min.

[0074] Amplification results are displayed in two ways:

[0075] (1) Agarose gel electrophoresis: The presence of ladder-shaped bands in the lane is considered positive, indicating the presence of a GABA receptor site mutation, such as Figure 1 If no ladder-shaped bands appear, it is judged as negative, indicating that no GABA receptor site mutation is found.

[0076] (2) Fluorescent dye colorimetric method: After amplification, place the product test tube on white paper for color observation. If the test tube turns sky blue, it is positive, indicating the presence of a GABA receptor site mutation, such as Figure 2 , no sky blue appears, which means negative, and there is no GABA receptor site mutation.

[0077] Example 4

[0078] LAMP detection of pear borer population in the field

[0079] The pear borer population DNA extracted in the process of Example 1 was used as a template, and the primer set designed in Example 1 was used as a detection primer to detect the pear borer GABA receptor site mutation using LAMP technology. The LAMP reaction system is as follows: 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 (7 μL is added after the inner and outer primers are mixed, the ratio of the total volume of the outer primers F3 and B3 to the total volume of the inner primers FIP and BIP is 8:1, the outer primers F3 and B3 are added in equal amounts, and the inner primers FIP and BIP are added in equal amounts), 1 μL of 10 μM each of the forward loop primer Loop-F and the reverse loop primer Loop-B is added, 2.5 μL of 10× LAMP PCR isothermal amplification reaction mixture, 1 μL of 8U Bst polymerase, 2.0 μL of DNA template (different dilution multiples), 5 μL of 5 mM betaine, and the volume is made up to 25 μL with sterile ultrapure water. Before amplification, 2 μL of 250 μM HNB colorimetric reagent, and finally add 25 μL paraffin oil to seal (to prevent aerosol contamination and avoid false positives), and water bath at 65°C for 60 min. The composition and content of the substance in the 2.5 μL LAMP PCR isothermal amplification reaction mixture are: dNTPs 10mmol, pH 8.8 Tris-HCl 20mmol, KCl 10mmol, MgSO42 mmol, (NH4) 2 SO 4 10 mmol, 0.1% Triton X-100.

[0080] After the amplification, 5 μL of the product was taken and detected by 1.0% agarose gel electrophoresis. The electrophoresis detection conditions were: voltage 80 V, current 400 A, and detection time 50 min.

[0081] The amplification results were displayed in two ways: (1) agarose gel electrophoresis: the appearance of ladder-shaped bands indicated the presence of GABA receptor site mutations, e.g. Figure 3 ; (2) Fluorescent dye colorimetric method: After amplification, if the product tube appears sky blue, it is positive, indicating the presence of a GABA receptor site mutation, such as Figure 4 .

[0082] Comparative Example 1:

[0083] Replacement of LAMP primers for detection

[0084] 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.

[0085] 1. Extraction of genomic DNA from adult single-headed pear borer

[0086] The genomic DNA of single-headed pear borer adult moths No. 1 to 6 was extracted according to the genomic DNA extraction method described in Example 2.

[0087] 2. LAMP reaction

[0088] In the LAMP reaction system, the mass concentrations of the outer primers F3-1 and B3-1 were both 10 μM, the mass concentrations of the inner primers FIP-1 and BIP-1 were both 40 μM (7 μL was added after the inner and outer primers were mixed, the volume ratio of the outer primers F3-1, B3-1 to the inner primers FIP-1, BIP-1 was 8:1, the outer primers F3-1 and the outer primers B3-1 were added in equal amounts, and the inner primers FIP-1 and the inner primers BIP-1 were added in equal amounts), 1 μL of the forward loop primer Loop-F1 and the reverse loop primer Loop-B1 were added at 10 μM each, 2.5 μL of 10× LAMP PCR isothermal amplification reaction mixture, 1 μL of 8U Bst polymerase, 1 μL of DNA template, 5 μL of 5mM betaine, and the volume was supplemented to 25 μL with sterile ultrapure water. Before amplification, 1 μL of 250 μM HNB colorimetric reagent, and finally add 25 μL paraffin oil to seal, and water bath at 65°C for 60 min. The material composition and content of the 2.5 μL AMP PCR isothermal amplification reaction mixture are: dNTPs 10mmol, pH 8.8 Tris-HCl 20mmol, KCl 10mmol, MgSO 4 2mmol, (NH4) 2 SO 4 10 mmol, 0.1% Triton X-100.

[0089] After the amplification, 5 μL of the product was taken and detected by 1.0% agarose gel electrophoresis. The electrophoresis detection conditions were: voltage 80 V, current 400 A, and detection time 50 min.

[0090] The specific primer sequences are as follows:

[0091] F3-1: 5'-GTCGTTCTGGCTGAACCG-3', SEQ ID NO: 8;

[0092] B3-1: 5'-TTGGCCATATATCCTACTGTAGC-3', SEQ ID NO: 9;

[0093] FIP-1:

[0094] 5'-GGTGGAGGACATGAGCGTTGT-ACGCCGGCGCCGTGTCGC-3', SEQ ID NO: 10;

[0095] BIP-1:

[0096] 5'-GCGCTGCCTAAGATCTCCTACG-AGACCATGACGAAACAGGTG-3', SEQ ID NO: 11;

[0097] Loop-F1: 5'-GGTGAGCACGGTGGTGA-3', SEQ ID NO: 12;

[0098] Loop-B1: 5'-TCAAGTCCATCGACGTCTACC-3', SEQ ID NO: 13.

[0099] Figure 5 and 6 The test results showed that pear borer individuals 1 to 2 were wild type, and tubes 1 to 2 did not change color. Peach borer individuals 3 to 5 contained GABA receptor A302S site mutations, but reaction tubes 3 to 5 did not change color either, and remained violet, indicating that normal LAMP reaction could not be performed after changing the LAMP primer sequence.

[0100] The change of LAMP detection primers (inner primers, outer primers and loop primers) makes the LAMP reaction unable to proceed. Therefore, in the present invention, primer design is a limiting factor, and inappropriate primer sequences will lead to the inability to perform correct LAMP detection, indicating that the primers provided by the present invention are specific.

[0101] The above description is for the preferred embodiment of the present invention, but should not be construed as limiting the present invention. Those skilled in the art may make other changes and modifications to these embodiments based on the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiment and all changes and modifications falling 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. 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 primer set for detecting GABA receptor mutations in 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, a forward loop primer Loop-F and a reverse loop primer Loop-B, and the sequence of the primer set is as follows: F3: 5'-CGTTCTGGCTGAACCGAA-3'; B3: 5'-TTGGCCATATATCCTACTGTAGC-3'; FIP: 5'- GGTGGAGGACATGAGCGTTGTACGCCGGCGCGCGTGTCGC-3'; BIP: 5'- GCGCTGCCTAAGATCTCCTACGAGACCATGACGAAACAGGTG-3'; Loop-F: 5'-TGAGCACGGTGGTGACG-3'; Loop-B: 5'-TCAAGTCCATCGACGTCTACC-3'; The LAMP detection primer set is used to detect the GABA receptor mutation of the fruit borer. The GABA receptor mutation sequence of the fruit borer is shown in SEQ ID NO:

7. The 844 bp of the sequence shown in SEQ ID NO: 7 has a G / t mutation.

2. A LAMP detection kit for GABA receptor mutation of fruit borer, characterized in that: Comprising the primer set according to claim 1.

3. Use of the primer set according to claim 1 or the kit according to claim 2 in detecting GABA receptor mutations in borer.

4. The use according to claim 3, characterized in that The primer set or the detection kit is used for detecting pear borer.

5. A method for detecting a GABA receptor mutation in pear borer, characterized in that: The following steps are involved: Extracting genomic DNA from the sample to be tested; Using the extracted DNA as a template, amplification is performed in a LAMP reaction system using the primer set described in claim 1; Determine the amplification results.

6. The LAMP detection method according to claim 5, characterized in that The reaction system includes: 10 μM each of outer primers F3 and B3, 40 μM each of inner primers FIP and BIP, 10 μM each of loop primers Loop-F and Loop-B, 2.5 μL of 10×LAMP reaction mixture, 1 μL of 8U Bst polymerase, 1 μL of DNA template, 5 μL of 5 mM betaine, ddH2O is added to 25 μL, and then 25 μL of paraffin oil is added to seal; Among them, the total amount of outer primers F3, B3 and inner primers FIP, BIP added was 7 μL, the total amount of loop primers Loop-F and Loop-B added was 1 μL, and the ratio of the total volume of outer primers F3, B3 to the total volume of inner primers FIP, BIP was 8:1, the volume ratios of outer primer F3 to outer primer B3, inner primer FIP to inner primer BIP, and loop primers Loop-F to Loop-B were all 1:

1.

7. The detection method according to claim 6, characterized in that: The composition and content of substances in 2.5 μL LAMP reaction mixture are: dNTPs 10 mmol, pH 8.8 Tris-HCl 20 mmol, KCl 10 mmol, MgSO4 2 mmol, (NH4)2SO4 10 mmol, 0.1% Triton X-100.

8. The detection method according to claim 6, characterized in that: Amplification conditions were incubated at 65°C for 50 min.

9. The detection method according to claim 6, characterized in that: The amplification results were determined using the following methods: Fluorescent dye colorimetric method: Add dye HNB to the amplified product for colorimetric reaction. If the reaction system turns blue, it indicates that the sample to be tested contains the GABA receptor mutation of the fruit borer; if the reaction system turns purple, it indicates that the sample to be tested does not contain the GABA receptor mutation of the fruit borer. or Agarose gel electrophoresis: If the amplified product shows a ladder-shaped amplification band, it indicates that the sample to be tested contains a GABA receptor mutation of the fruit borer. If no characteristic ladder-shaped amplification bands appear, it indicates that the sample to be tested does not contain the GABA receptor mutation of the fruit borer.

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