Method and special kit for rapidly detecting sodium ion channel L1014F mutation of Liriomyza trifoliata

Abstract

The invention discloses a method for rapidly detecting sodium ion channel L1014F mutation of liriomyza trifoliata and a special kit, and belongs to the technical field of agricultural biology. The method comprises the following steps: extracting the DNA of the liriomyza trifoliate; carrying out PCR (Polymerase Chain Reaction) amplification by adopting two groups of primer pairs; an upstream primer of the first group of primer pair is as shown in SEQ ID NO.2, an upstream primer of the second group of primer pair is as shown in SEQ ID NO.1, and a downstream primer of the second group of primer pair is as shown in SEQ ID NO.3. Result judgment: when a 502 bp band appears in amplification of the first group of primer pairs, the L1014F site of the sample is not mutated; and when a 502 bp band appears in the amplification of the second group of primer pairs, the L1014F site of the sample is mutated. According to the method, mononucleotide variation typing can be achieved only through a conventional PCR instrument and electrophoresis, operation is easy and convenient, and the method is suitable for rapid monitoring of pyrethroid drug resistance of liriomyza trifoliate and investigation of field population resistance gene frequency.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a method and a dedicated reagent kit for rapid detection of the L1014F mutation in the sodium ion channel of the leafminer fly. Background Technology

[0002] The three-leaf leafminer (Liriomyza trifolii) is a major agricultural pest belonging to the family Liriomyzae in the order Diptera, widely distributed in tropical and temperate regions worldwide. The larvae feed on leaf tissue, creating tunnels that cause leaf withering and even plant death. Adults can transmit various pathogens, including tomato leaf spot virus, resulting in indirect losses of 3-5 times the direct yield. Its hosts include over 150 species of plants from 32 families, including Solanaceae, Cucurbitaceae, and Fabaceae, severely damaging economic crops such as cucumbers, tomatoes, and cowpeas. The adult leafminer is only 1.3–2.3 mm long and morphologically highly similar to closely related species, leading to an identification error rate exceeding 40%.

[0003] Pyrethroid insecticides are widely used due to their high efficacy and low toxicity, but long-term, high-dose application has induced resistance in various insects. Their target is voltage-sensitive sodium ion channels, and knockdown resistance (kDR) is the most important mechanism for resistance development in this class of insecticides. The resistance phenotype is closely linked to mutations in sodium ion channels. Previous research has shown that a CTT to TTT point mutation occurs in the coding codon at amino acid 1018 (corresponding to the commonly used name L1014F) of the S6 region of the second domain of the sodium ion channel gene in field populations of the leafminer (GenBank accession number: MT670290), resulting in a change from leucine (Leu) to phenylalanine (Phe), i.e., the L1014F mutation. This mutation is the key molecular mechanism leading to high-level knockdown resistance in leafminer to pyrethroid insecticides. In some field populations in Hainan, the mutation frequency has reached 100%, indicating an extremely severe resistance situation. Currently, there is limited research in China on pyrethroid resistance in the leafminer fly, and effective and rapid monitoring methods for its resistance mutation sites are lacking. Therefore, it is urgent to develop a specific method and dedicated primers for detecting the L1014F mutation in the leafminer fly to achieve rapid monitoring of its pyrethroid resistance.

[0004] Current technologies for monitoring antibiotic resistance in the trifoliate leafminer mainly include bioassays, PCR-sequencing, and RNA interference. Bioassays are time-consuming and have low sensitivity; while PCR-sequencing is fast and accurate, it still lacks specific primers and standardized detection procedures for the L1014F site; RNAi technology is mainly used for gene function verification and has not yet been translated into a means of resistance monitoring.

[0005] Existing technologies have the following problems: First, drug resistance is a prominent issue. Long-term use of a single drug leads to an exponential increase in resistance, and the efficacy of conventional doses is less than 40%, forming a vicious cycle of "increasing dose - increasing resistance." Second, pests are covert infestations. Larvae feed on leaf tissue, and adults are tiny, making it difficult to achieve early warning through conventional monitoring. Third, chemical agents have a kill rate of over 80% against natural enemies, disrupting ecological pest control mechanisms and significantly shortening the resurgence cycle of pests. Fourth, molecular detection technology is lacking. There is currently no specific detection system for the L1014F mutation site of the three-leaf leafminer, which cannot meet the needs of accurate monitoring.

[0006] Current control of the leafminer fly faces challenges such as insufficient identification accuracy and a lack of assessment methods, lagging morphological identification, and a lack of highly sensitive molecular detection tools for the L1014F resistance mutation. Therefore, there is an urgent need to develop a molecular detection technology and kit based on highly specific primers to accurately amplify target gene fragments from complex samples, rapidly identify the L1014F resistance mutation, and provide technical support for resistance management and evaluation of biological control effectiveness. Summary of the Invention

[0007] The purpose of this invention is to provide a method and specific primers for identifying sodium ion channel gene mutations in *S. trifoliata*, particularly a method and specific kit for identifying the resistance-related mutation site L1014F in *S. trifoliata*, for monitoring resistance of *S. trifoliata* to pyrethroid insecticides, thereby solving the problems mentioned in the background art.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for rapid detection of the L1014F sodium channel mutation in the leafminer fly includes the following steps:

[0010] 1) Extract DNA from the trifoliate leafminer;

[0011] 2) Two sets of primer pairs were used for PCR amplification, and the primer pair sequences are as follows:

[0012] First primer pair: Upstream primer 1: 5'-CCACAGTTGTGATTGGAACC-3'.

[0013] Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3';

[0014] Second primer pair: Upstream primer 2: 5'-CCACAGTTGTGATTGGAACT-3',

[0015] Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3';

[0016] 3) Result interpretation: If the first primer pair amplifies a 502 bp band, the sodium ion channel gene mutation site L1014F of the tested *Leymus trifoliata* sample has not mutated; if the second primer pair amplifies a 502 bp band, the sodium ion channel gene mutation site L1014F of the tested *Leymus trifoliata* sample has mutated, and if no band appears, the tested *Leymus trifoliata* has not mutated.

[0017] In some embodiments, in step 1), DNA from the trifoliate leafminer fly is extracted using a DNA extraction kit.

[0018] In some embodiments, in step 2), the 25 μL PCR amplification system consists of: 1 μL template DNA, 1 μL 10M upstream primer, 1 μL 10M downstream primer, 12.5 μL Green Taq Mix, and 9.5 μL ddH2O.

[0019] In some embodiments, in step 2), the PCR amplification conditions are: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 53°C annealing for 15 s, 72°C extension for 30 s, for 35 cycles; 72°C extension for 5 min.

[0020] In some embodiments, the method for rapid detection of the L1014F sodium channel mutation in the leafminer fly includes the following steps:

[0021] 1) Genomic DNA of the trifoliate leafminer was extracted using a DNA extraction kit;

[0022] 2) Using the genomic DNA obtained in step 1) as a template, allele-specific PCR amplification was performed using two sets of primer pairs; among which,

[0023] First primer pair: Upstream primer 1: 5'-CCACAGTTGTGATTGGAACC-3'.

[0024] Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3';

[0025] Second primer pair: Upstream primer 2: 5'-CCACAGTTGTGATTGGAACT-3',

[0026] Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3';

[0027] The 25 μL PCR amplification system consisted of: 1 μL template DNA, 1 μL 10M upstream primer, 1 μL 10M downstream primer, 12.5 μL Green Taq Mix, and 9.5 μL ddH2O.

[0028] PCR amplification conditions: 95℃ pre-denaturation for 3-5 min; 95℃ denaturation for 10-30 s, 54℃-55℃ annealing for 15 s, 72℃ extension for 30-90 s, for 35 cycles;

[0029] 3) Result interpretation: If the first primer pair amplifies a 502 bp band, then the sodium ion channel gene mutation site L1014F of the tested *Tectus trifoliata* sample has not mutated; if the second primer pair amplifies a 502 bp band, then the sodium ion channel gene mutation site L1014F of the tested *Tectus trifoliata* sample has mutated.

[0030] The dedicated kit for the rapid detection of the L1014F sodium channel mutation in the leafminer fly includes at least one dose of two primer pair solutions; the sequences of the primer pairs are as follows:

[0031] First primer pair: Upstream primer 1: 5'-CCACAGTTGTGATTGGAACC-3'.

[0032] Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3';

[0033] Second primer pair: Upstream primer 2: 5'-CCACAGTTGTGATTGGAACT-3',

[0034] Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3'.

[0035] In some embodiments, the dedicated kit further includes a single-use or higher amount of trifoliate leafminer genomic DNA extraction solution and necessary solutions for the PCR system.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention provides a method for detecting the L1014F mutation in *Leymus trilobus* that requires no sequencing, no quantitative fluorescence, and no high-resolution melting curve. It only requires a conventional PCR instrument and electrophoresis equipment to achieve accurate single-nucleotide variant typing, making it suitable for rapid screening in grassroots laboratories and fields. This invention uses two sets of allele-specific primers for parallel amplification, directly identifying wild-type homozygous, mutant homozygous, and heterozygous genotypes by the presence or absence of amplified bands, completely eliminating dependence on sequencing platforms. The primer pairs have high site and species specificity, producing a 502 bp target band only in either the mutant or wild-type *Leymus trilobus* template. The method is simple to operate, provides intuitive results, and is low in cost.

[0038] This invention solves the technical bottlenecks of existing PCR-sequencing methods, which rely on large instruments, have long detection cycles, and cannot achieve rapid on-site screening. It provides an efficient, convenient, and easily promoted molecular detection tool for population monitoring of pyrethroid resistance in the leafminer fly, investigation of resistance gene frequencies, and precision pesticide application in the field. Attached Figure Description

[0039] Figure 1 This is a flowchart of the detection process in Example 1;

[0040] Figure 2 This is an electrophoresis diagram of the PCR reaction performed on DNA extracted from the mutant trifoliate leafminer in Example 1; in the diagram: lane M is the DNA Marker 2000 band, lanes 1-3 are the bands of the mutant primer amplification product; lanes 4-6 are the bands of the CK primer amplification product;

[0041] Figure 3 This is a sequencing diagram of the L1014F site of the sodium ion channel gene in *Tectus trifoliata*, from Example 1 of a rapid detection method for verifying the mutation of the L1014F mutation in the sodium ion channel gene of *Tectus trifoliata*.

[0042] Figure 4 The diagram shows the results of the interspecies specificity experiment; lanes 1 and 2 are for the trifoliate leafminer, lanes 3 and 4 are for the American leafminer, lanes 5 and 6 are for the South American leafminer, and M is the D2000 marker;

[0043] Figure 5 The image shows the template sensitivity test results; lane M is the DNA Marker 2000 band; lane 1 is a single *S. trifoliata* sample, lane 2 is a sample of 2 *S. trifoliata*, lane 3 is a sample of 3 *S. trifoliata*, lane 4 is a sample of 4 *S. trifoliata*, and lane 5 is a sample of 5 *S. trifoliata*.

[0044] Figure 6 The image shows the results of the detection limit experiment for the primer pair of *S. trifoliata*. Lane 1 shows the PCR amplification band of a single *S. trifoliata* diluted 2-fold; lane 2 shows the PCR amplification band of a single *S. trifoliata* diluted 5-fold; lane 3 shows the PCR amplification band of a single *S. trifoliata* diluted 10-fold; lane 4 shows the PCR amplification band of a single *S. trifoliata* diluted 20-fold; lane 5 shows the PCR amplification band of a single *S. trifoliata* diluted 50-fold; and lane 6 shows the PCR amplification band of a single *S. trifoliata* diluted 100-fold.

[0045] Figure 7 Image showing the L1014F mutation detection results of 8 leafminer flies in Yazhou District, Sanya City, Hainan Province. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0047] Example 1

[0048] The effect of leafminer-specific primers on the amplification of leafminer DNA

[0049] The testing flowchart is as follows Figure 1 As shown, the steps are as follows:

[0050] 1. Field population sampling of the leafminer fly (Leymus trifoliata).

[0051] Leaves with leaf miner tunnels were collected from cowpea fields, placed in plastic bags, and taken back to the laboratory. After dissecting the tunnels with a dissecting needle, the larvae were gently swept into centrifuge tubes with a small brush.

[0052] 2. DNA extraction from a single individual of the trifoliate leafminer fly.

[0053] DNA was extracted from the trifoliate leafminer fly using the TransDirect Animal Tissue PCR Kit from Beijing TransGen Biotechnology Co., Ltd., following the instructions. The extracted DNA was stored at -20℃ for later use.

[0054] 3. PCR amplification of DNA using primers.

[0055] A single-headed leafminer fly was placed in a 1.5 mL centrifuge tube and ground to extract the DNA. The obtained DNA solution was stored at -20℃ for later use. 2 μL of the solution was used as a DNA template for PCR amplification.

[0056] (1) Synthesize specific primers for detecting leafminer flies.

[0057] The specific primer sequences for leafminer fly are as follows:

[0058] Primer LT1014: 5'-CCACAGTTGTGATTGGAACT-3' (SEQ ID NO.1);

[0059] Primer LT-CK: 5'-CCACAGTTGTGATTGGAACC-3' (SEQ ID NO.2);

[0060] Primer LT-R: 5'-CCTCTATGGTTTAGCTCATG-3' (SEQ ID NO.3).

[0061] (2) PCR amplification

[0062] The reaction system consisted of 25 μL of GreenTaq Mix enzyme, 1 μL of DNA template, 1 μL of each primer (LT1014 and LT-CK were used in combination with primer LT-R), and double-distilled water was added to bring the total volume to 25 μL.

[0063] PCR amplification program: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles.

[0064] 4. Identification of PCR products

[0065] PCR amplification products were separated by 1% agarose gel electrophoresis. After ethidium bromide staining, the products were analyzed using a gel imaging system with the D2000 standard molecular weight as a reference, and the size of the amplified products was used to determine their molecular weight. The electrophoresis results are shown in Table 1. The electrophoresis results for primer pairs LT1014 and LT-R amplification of the mutant trifoliate leafminer are shown in Table 1. Figure 2 As shown.

[0066] Table 1 Comparison of Detection Results

[0067]

[0068] 5. Purification and recovery of PCR products

[0069] PCR products were purified and recovered using a PCR product purification and recovery kit. The PCR product purification and recovery procedure was as follows: Determine the volume of the PCR product using a pipette, add 5 volumes of CP Buffer; mix thoroughly by pipetting, transfer to a centrifuge column, centrifuge at 13000 rpm for 1 min, and discard the waste liquid; add 700 L of DNA wash Buffer, centrifuge at 13000 rpm for 1 min, discard the waste liquid, and repeat once; after centrifuging at 13000 rpm for 2 min, add 30-50 L of Elution Buffer to the white portion in the middle of the centrifuge column, connect a 1.5 mL centrifuge tube, incubate at room temperature for 2 min, and centrifuge at 13000 rpm for 1 min. Take 20-30 μL for sequencing.

[0070] 6. Validate the recovered products by sequencing using LT-R.

[0071] The PCR products with bright bands were sent to Beijing Qingke Biotechnology Co., Ltd. for reverse sequencing using sequencing primer LT-R. The sequencing results showed that the product sequence obtained by primer pairs LT-CK and LT-R amplification of wild-type *Leymus trilobus* is shown in SEQ ID NO.4, and the product sequence obtained by primer pairs LT1014 and LT-R amplification of mutant *Leymus trilobus* is shown in SEQ ID NO.5. The DNA coding sequence at position L1014F of the *Leymus trilobus* sodium ion channel gene was mutated from CTT to TTT, resulting in a change from leucine to phenylalanine at this site. Figure 3 As shown. Sequencing verification results indicate that the dedicated primers provided in this invention can effectively identify the mutation (L1014F mutation) in the sodium ion channel gene related to resistance in the trifoliate leafminer.

[0072] Example 2

[0073] Amplification of different species of L1014F drug-resistant mutant leafminer flies using primer pairs

[0074] 1. Preparation of Genomic DNA from Leafminer Fly

[0075] Genomic DNA was extracted using the TransDirect Animal Tissue PCR Kit from Beijing TransGen Biotechnology Co., Ltd. 3 μL of AD2 buffer was added to 12 μL of AD1 buffer and mixed thoroughly. Then, single-headed leafminer bodies were thoroughly ground in 15 μL of the AD1 and AD2 premix. After incubation at room temperature for 10 min, the mixture was incubated at 95°C for 3 min. Finally, 12 μL of AD3 buffer was added and mixed thoroughly with the incubated sample solution. The mixture was then stored at -20°C.

[0076] 2. Synthesis of specific primers for detecting the trifoliate leafminer.

[0077] Primer LT1014: 5'-CCACAGTTGTGATTGGAACT-3';

[0078] Primer LT-CK: 5'-CCACAGTTGTGATTGGAACC-3';

[0079] Primer LT-R: 5'-CCTCTATGGTTTAGCTCATG-3'.

[0080] The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0081] 3. PCR amplification procedure

[0082] Pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 55℃ for 30 s, and extension at 72℃ for 30 s, for a total of 35 cycles.

[0083] 4. Identification of PCR products

[0084] Take 2 μL of PCR amplification product, separate it by 1% agarose gel electrophoresis, stain it with nucleic acid dye, and then analyze it on a gel imaging system.

[0085] In this system, the determination is based on the presence or absence of amplified products. The D2000 standard molecular weight is used as a reference.

[0086] 5. Results

[0087] PCR amplification was performed using primers with genomic DNA from *Leymus chinensis*, *Leymus chinensis*, and *Leymus chinensis* as templates, respectively. The results are as follows: Figure 4 The results showed that only the trifoliate leafminer could amplify the band, indicating that the primer was highly specific and could only amplify the corresponding fragment of the trifoliate leafminer, while having no amplification effect on other closely related species.

[0088] Example 3

[0089] Sensitivity test of primers against L1014F drug-resistant mutant leafminer genomic DNA template

[0090] 1. Preparation of Genomic DNA from Leafminer Fly

[0091] Genomic DNA was extracted from leafminer flies using the TransDirect Animal Tissue PCR Kit from Beijing TransGen Biotechnology Co., Ltd. 3 μL of AD2 buffer was added to 12 μL of AD1 buffer and mixed thoroughly. Leafminer flies were then thoroughly ground in 15 μL of the AD1 and AD2 premix to prepare five genomic DNA templates. DNA was extracted from 1, 2, 3, 4, and 5 live leafminer flies, respectively. After incubation at room temperature for 10 min, the samples were incubated at 95°C for 3 min. Then, 12 μL of AD3 buffer was added and mixed thoroughly with the incubated samples. The samples were stored at -20°C.

[0092] 2. Synthesis of specific primers for detecting the trifoliate leafminer.

[0093] Primer LT1014: 5'-CCACAGTTGTGATTGGAACT-3';

[0094] Primer LT-CK: 5'-CCACAGTTGTGATTGGAACC-3';

[0095] Primer LT-R: 5'-CCTCTATGGTTTAGCTCATG-3'.

[0096] Synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0097] 3. PCR amplification procedure

[0098] Pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 55℃ for 30 s, and extension at 72℃ for 30 s, for a total of 35 cycles.

[0099] 4. Identification of PCR products

[0100] Take 2 μL of PCR amplification product, separate it by 1% agarose gel electrophoresis, stain it with nucleic acid dye, and then analyze it on a gel imaging system.

[0101] In this system, the size of the amplified product is used for determination. The D2000 standard molecular weight is used as a reference.

[0102] 5. Results

[0103] Using primers, PCR amplification was performed using genomic DNA from 1, 2, 3, 4, and 5 leafminers, respectively, as templates. The results showed that ( Figure 5 Using the genomic DNA of a single-headed leafminer fly as a template, a clear 502 bp specific band (lane 1) can be amplified, and the target band can be amplified in the other 4 groups as well, indicating that the primers of the present invention have high template sensitivity and can meet the needs of micro-detection of single-headed flies.

[0104] Example 4

[0105] Detection limit of primer pairs for L1014F drug-resistant mutant leafminer genomic DNA template

[0106] 1. Preparation of Genomic DNA from Leafminer Fly

[0107] Genomic DNA was extracted from *Leymus chinensis* using the TransDirect Animal Tissue PCR Kit from Beijing TransGen Biotechnology Co., Ltd. 3 μL of AD2 buffer was added to 12 μL of AD1 buffer and mixed thoroughly. Single *Leymus chinensis* cells were then thoroughly ground in 15 μL of the AD1 and AD2 premix, incubated at room temperature for 10 min, followed by incubation at 95°C for 3 min. 12 μL of AD3 buffer was then added and mixed thoroughly with the incubated sample. The initial concentration was 24.472 ng / μL, measured spectrophotometer. The template was diluted 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, and 1000-fold with ultrapure water and stored at -20°C.

[0108] 2. Synthesis of specific primers for detecting the trifoliate leafminer.

[0109] Primer LT1014: 5'-CCACAGTTGTGATTGGAACT-3';

[0110] Primer LT-CK: 5'-CCACAGTTGTGATTGGAACC-3';

[0111] Primer LT-R: 5'-CCTCTATGGTTTAGCTCATG-3'.

[0112] The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0113] 3. PCR amplification

[0114] The reaction system consisted of 25 μL of the following components: 12.5 μL of GreenTaq Mix enzyme, 1 μL of DNA template, 1 μL each of forward and reverse primers (10 μM), and 9.5 μL of ddH2O.

[0115] Pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 55℃ for 30 s, and extension at 72℃ for 30 s, for a total of 35 cycles.

[0116] 4. Identification of PCR products

[0117] Take 2 μL of PCR amplification product, separate it by 1% agarose gel electrophoresis, stain it with nucleic acid dye, and determine the presence or absence of amplification product in a gel imaging system. Use D2000 standard molecular weight as a reference.

[0118] 5. Results

[0119] PCR amplification was performed using primers with genomic DNA templates diluted 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, and 1000-fold as templates from a single-headed leafminer. The results showed that ( Figure 6 The target band could be amplified from genomic DNA templates diluted 10, 20, 50, 100, 500, and 1000 times. However, non-specific amplification began to appear after dilution of 500 times, and the band brightness decreased with increasing dilution concentration. Therefore, this experiment shows that the detection limit of this primer is at least 0.049 ng / μL.

[0120] Example 5

[0121] The mutation status of sodium ion channel L1014F in a field population of leafminer fly collected in Sanya City, Hainan Province in 2026 was detected.

[0122] 1. Preparation of genomic DNA from the trifoliate leafminer fly

[0123] Genomic DNA was extracted from leafminer flies using the TransDirect Animal Tissue PCR Kit from Beijing TransGen Biotechnology Co., Ltd. 3 μL of AD2 buffer was added to 12 μL of AD1 buffer and mixed thoroughly. Then, a single leafminer fly was thoroughly ground in 15 μL of the AD1 and AD2 premix. After incubation at room temperature for 10 min, it was incubated at 95°C for 3 min. Finally, 12 μL of AD3 buffer was added and mixed thoroughly with the incubated sample solution. The mixture was then stored at -20°C.

[0124] 2. Synthesis of specific primers for detecting the trifoliate leafminer.

[0125] Primer LT1014: 5'-CCACAGTTGTGATTGGAACT-3';

[0126] Primer LT-CK: 5'-CCACAGTTGTGATTGGAACC-3';

[0127] Primer LT-R: 5'-CCTCTATGGTTTAGCTCATG-3'.

[0128] The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0129] 3. PCR amplification

[0130] The reaction system consisted of 25 μL of the following components: 12.5 μL of GreenTaq Mix enzyme, 1 μL of DNA template, 1 μL each of forward and reverse primers (10 μM), and 9.5 μL of ddH2O.

[0131] Pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 10 s, annealing at 53℃ for 30 s, and extension at 72℃ for 90 s, for a total of 35 cycles.

[0132] 4. Identification of PCR products

[0133] Take 2 μL of PCR amplification product, separate it by 1% agarose gel electrophoresis, stain it with nucleic acid dye, and determine the size of the amplification product in a gel imaging system. Use the D2000 standard molecular weight as a reference.

[0134] 5. Results

[0135] Using primers LT1014 and LT-R, PCR amplification was performed using *Tectus trifoliata* DNA as a template. The results are as follows: Figure 7 As shown, among the eight leafminer samples collected in Sanya City, samples 1-8 were all leafminers with the L1014F mutation, meaning that the frequency of the L1014F drug resistance mutation in the leafminer population in Yazhou District, Sanya City, Hainan Province was 100% in this test.

[0136] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A method for rapid detection of the L1014F sodium ion channel mutation in the leafminer fly, characterized in that, Includes the following steps: 1) Extract DNA from the trifoliate leafminer; 2) Two sets of primer pairs were used for PCR amplification, and the primer pair sequences are as follows: First primer pair: Upstream primer 1: 5'-CCACAGTTGTGATTGGAACC-3'. Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3'; Second primer pair: Upstream primer 2: 5'-CCACAGTTGTGATTGGAACT-3', Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3'; 3) Result interpretation: If the first primer pair amplifies a 502 bp band, the sodium ion channel gene mutation site L1014F of the tested *Leymus trifoliata* sample has not mutated; if the second primer pair amplifies a 502 bp band, the sodium ion channel gene mutation site L1014F of the tested *Leymus trifoliata* sample has mutated, and if no band appears, the tested *Leymus trifoliata* has not mutated.

2. The method for rapid detection of the L1014F sodium ion channel mutation in *Leymus trifoliata* according to claim 1, characterized in that, In step 1), DNA was extracted from the trifoliate leafminer using a DNA extraction kit.

3. The method for rapid detection of the L1014F sodium ion channel mutation in *Tectus trifoliata* according to claim 1, characterized in that, In step 2), the 25 μL PCR amplification system consists of: 1 μL template DNA, 1 μL 10M upstream primer, 1 μL 10M downstream primer, 12.5 μL Green Taq Mix, and 9.5 μL ddH2O.

4. The method for rapid detection of the L1014F sodium ion channel mutation in *Leymus trifoliata* according to claim 1, characterized in that, In step 2), the PCR amplification conditions are as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 53℃ annealing for 15 s, 72℃ extension for 30 s, for 35 cycles; 72℃ extension for 5 min.

5. The method for rapid detection of the L1014F sodium ion channel mutation in *Tectus trifoliata* according to claim 1, characterized in that, Includes the following steps: 1) Genomic DNA of the trifoliate leafminer was extracted using a DNA extraction kit; 2) Using the genomic DNA obtained in step 1) as a template, allele-specific PCR amplification was performed using two sets of primer pairs; among which, First primer pair: Upstream primer 1: 5'-CCACAGTTGTGATTGGAACC-3'. Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3'; Second primer pair: Upstream primer 2: 5'-CCACAGTTGTGATTGGAACT-3', Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3'; The 25 μL PCR amplification system consisted of: 1 μL template DNA, 1 μL 10M upstream primer, 1 μL 10M downstream primer, 12.5 μL Green Taq Mix, and 9.5 μL ddH2O. PCR amplification conditions: 95℃ pre-denaturation for 3-5 min; 95℃ denaturation for 10-30 s, 54℃-55℃ annealing for 15 s, 72℃ extension for 30-90 s, for 35 cycles; 3) Result interpretation: If the first primer pair amplifies a 502 bp band, the sodium ion channel gene mutation site L1014F of the tested *Leymus trifoliata* sample has not mutated; if the second primer pair amplifies a 502 bp band, the sodium ion channel gene mutation site L1014F of the tested *Leymus trifoliata* sample has mutated, and if no band appears, the tested *Leymus trifoliata* has not mutated.

6. A dedicated kit for the rapid detection method of the L1014F sodium ion channel mutation in *Tectus trifoliata* as described in claim 1, characterized in that, It includes at least two sets of primer pair solutions for a single application; the sequences of the primer pairs are as follows: First primer pair: Upstream primer 1: 5'-CCACAGTTGTGATTGGAACC-3'. Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3'; Second primer pair: Upstream primer 2: 5'-CCACAGTTGTGATTGGAACT-3', Downstream primer: 5'-CCTCTATGGTTTAGCTCATG-3'.

7. The dedicated reagent kit according to claim 6, characterized in that, It also includes a single or multiple dose of Trifoliate Leafminer genomic DNA extraction solution and necessary solutions for the PCR system.

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