KASP primers for detecting drug resistance mutation of thrips and application of KASP primers
By designing a KASP primer combination targeting thrips, the problem of difficulty in detecting thrips resistance in existing technologies was solved, and a rapid, low-cost, high-throughput detection method was achieved, which is suitable for resistance detection of multiple populations.
Patent Information
- Application Number
- CN202410248140.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-05
- Publication Date
- 2025-09-05
AI Technical Summary
Existing technologies make it difficult to quickly, cost-effectively, and high-throughput detect thrips' resistance to insecticides. Traditional methods require live testing and are labor-intensive, while PCR amplification and sequencing methods have long detection cycles and high costs.
A KASP primer combination for thrips was designed to detect drug resistance-related SNP variant sites on genes such as sodium ion channels and GABA receptors, and combined with KASP technology for high-throughput, low-cost molecular detection.
It has achieved rapid, accurate, low-cost and high-throughput detection of thrips resistance, which is suitable for simultaneous detection of multiple populations and inference of the resistance level of populations to different insecticides.
Smart Images
Figure CN120591409A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of agricultural biotechnology and relates to rapid molecular detection of thrips drug resistance-related mutations. Background Art
[0002] Thrips are small insects of the order Thysanoptera. Many species are important agricultural pests, such as the melon thrips. Thrips palmi , Western Flower Thrips Frankliniella occidentalis , Thrips leucophylla Megalurothripsusitatus Each thrips species can infect dozens or even hundreds of host plants, including vegetables like eggplant, peppers, cucumbers, cantaloupes, and cowpeas, as well as a variety of fruit trees and flowers, causing significant economic losses to agricultural production. Due to the small size, hidden nature of thrips pests, short generation cycles, and high fecundity, effective prevention and control measures are lacking. Currently, farmers primarily use chemical agents for prevention and control, resulting in many thrips developing resistance to insecticides, including pyrethroids, spinosads, and the new insecticide bromidron. Previous studies have shown significant differences in insecticide resistance between different thrips species and within different populations of the same thrips species. Therefore, it is necessary to test for insecticide resistance in different thrips species and populations to facilitate the scientific and rational selection of effective insecticides.
[0003] Traditional methods for detecting insecticide resistance in thrips rely primarily on bioassays. While reliable, these methods require live specimens, placing high demands on sample collection, transportation, and husbandry. Furthermore, they are slow and labor-intensive, making them unsuitable for testing multiple populations simultaneously. Methods that determine the presence of resistance mutations by amplifying target genes through PCR, sequencing, and sequence comparison do not require live specimens and can test multiple populations simultaneously, but they still present challenges such as long testing cycles and high costs. KASP (competitive allele-specific PCR) utilizes fluorescent markers to accurately detect SNPs (single nucleotide polymorphisms) in sample DNA. Compared to PCR amplification and sequencing, KASP enables high-throughput, low-cost detection of resistance mutations. However, primers specifically designed for KASP detection of insecticide resistance in thrips are currently unavailable. Therefore, it is necessary to develop a KASP primer and method to detect the resistance mutation sites M918TAG, T929I, L1014FGT, T929C, A355S, V386I, G275V and I1042M on the thistle gene for monitoring the frequency of resistance mutations in field thrips. Summary of the Invention
[0004] The main purpose of the present invention is to achieve high-throughput rapid molecular detection of thrips resistance to pesticides.
[0005] To achieve the purpose of the present invention, three sets of KASP primer combinations are designed for detecting SNP variation sites associated with drug resistance on the sodium ion channel (kdr) gene of thrips cucurbitae; three sets of KASP primer combinations are designed for detecting SNP variation sites associated with drug resistance on the sodium ion channel (kdr) gene of thrips occidentalis; two sets of KASP primer combinations are designed for detecting SNP variation sites associated with drug resistance on the GABA receptor RDL subunit (RDL) gene of thrips cucurbitae; and two sets of KASP primer combinations are designed for detecting SNP variation sites associated with drug resistance on the GABA receptor RDL subunit (RDL) gene of thrips cucurbitae. ) gene associated with insecticide resistance; two sets of KASP primer combinations that can be used to detect SNP variant sites associated with insecticide resistance in the GABA receptor RDL subunit (RDL) gene of western flower thrips; one set of KASP primer combinations that can be used to detect SNP variant sites associated with insecticide resistance in the nicotinic acetylcholine receptor (nAChR) gene of flower thrips; one set of KASP primer combinations that can be used to detect SNP variant sites associated with insecticide resistance in the chitin synthase (CHS1) gene of western flower thrips;
[0006] Furthermore, the detection primers include a primer combination for detecting the 918th mutation Tpal_M918TAG, the 929th mutation Tpal_T929I and the 1014th mutation Tpal_L1014FGT of the sodium ion channel (kdr) protein sequence of melon thrips; a primer combination for detecting the 929th mutation Focc_T929I, Focc_T929C and the 1014th mutation FoccL_1014FGT of the sodium ion channel (kdr) protein sequence of western flower thrips; a primer combination for detecting the 355th mutation of the GABA receptor RDL subunit (RDL) protein sequence of bean thrips Primer combination for detecting the mutations Musi_A355S and Focc_V386I at position 386; primer combination for detecting the mutations Tpal_A355S and Tpal_V386I at position 355 of the GABA receptor RDL subunit (RDL) protein sequence of thrips citriodora; primer combination for detecting the mutations Focc_A355S and Focc_V386I at position 386 of the GABA receptor RDL subunit (RDL) protein sequence of thrips occidentalis; primer combination for detecting the mutation Fint_G275V at position 275 of the nicotinic acetylcholine receptor (nAChR) protein sequence of thrips occidentalis; primer combination for detecting the mutation Focc_I1042M at position 1042 of the chitin synthase (CHS1) protein sequence of thrips occidentalis.
[0007] Furthermore, the sequences of the two upstream primers included in the primer combination Tpal_M918TAG are shown in SEQ ID NO.: 1 and SEQ ID NO.: 2, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 3; the sequences of the two upstream primers included in the primer combination Tpal_T929I are shown in SEQ ID NO.: 4 and SEQ ID NO.: 5, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 6; the sequences of the two upstream primers included in the primer combination Tpal_L1014FGT are shown in SEQ ID NO.: 7 and SEQ ID NO.: 8, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 9; the sequences of the two upstream primers included in the primer combination Focc_T929I are shown in SEQ ID NO.: 10 and SEQ ID NO.: 11, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 12 : SEQ ID NO.: 13; the sequences of the two upstream primers included in the primer combination Focc_T929C are shown in SEQ ID NO.: 13 and SEQ ID NO.: 14, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 15; the sequences of the two upstream primers included in the primer combination Focc_L1014FGT are shown in SEQ ID NO.: 16 and SEQ ID NO.: 17, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 18; the sequences of the two upstream primers included in the primer combination Musi_A355S are shown in SEQ ID NO.: 19 and SEQ ID NO.: 20, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 21; the sequences of the two upstream primers included in the primer combination Tpal_A355S are shown in SEQ ID NO.: 22 and SEQ ID NO.: 23, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 24 The sequences of the two upstream primers included in the primer combination Focc_A355S are shown in SEQ ID NOs.: 25 and 26, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 27; the sequences of the two upstream primers included in the primer combination Musi_V386I are shown in SEQ ID NOs.: 28 and 29, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 30; the sequences of the two upstream primers included in the primer combination Tpal_V386I are shown in SEQ ID NOs.: 31 and 32, respectively.: 32, and the sequence of the downstream primer is shown in SEQ ID NO.: 33; the sequences of the two upstream primers contained in the primer combination Focc_V386I are shown in SEQ ID NO.: 34 and SEQ ID NO.: 35, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 36; the sequences of the two upstream primers contained in the primer combination Fint_G275V are shown in SEQ ID NO.: 37 and SEQ ID NO.: 38, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 39; the sequences of the two upstream primers contained in the primer combination Focc_I1042M are shown in SEQ ID NO.: 40 and SEQ ID NO.: 41, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 42.
[0008] Furthermore, the sequences of the two upstream primers of each primer combination are respectively connected to different fluorescent modified tags, which are used to distinguish different alleles during KASP typing;
[0009] The present invention also establishes a high-throughput rapid molecular detection method for thrips resistance, and the specific steps are as follows:
[0010] (1) The KASP primer combination Tpal_M918TAG was used to perform KASP reaction and SNP typing using the DNA of melon thrips as a template. If the fluorescence signal data of the amplified product was typed as G:G, it was a homozygous resistant individual; if the fluorescence signal data was typed as A:A, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele G in the tested population was statistically analyzed to infer the resistance level of the melon thrips population to cypermethrin (pyrethroid) insecticide.
[0011] (2) KASP primer combination Tpal_T929I was used to perform KASP reaction and SNP typing using the DNA of thrips as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as C:C, it was a homozygous sensitive individual; if the fluorescence signal data was typed as C:T, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was statistically analyzed to infer the resistance level of the thrips population to cypermethrin (pyrethroid) insecticide.
[0012] (3) KASP primer combination Tpal_L1014FGT was used to perform KASP reaction and SNP typing using the DNA of thrips as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:T, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was statistically analyzed to infer the resistance level of the thrips population to cypermethrin (pyrethroid) insecticide.
[0013] (4) KASP primer combination Focc_T929I was used to perform KASP reaction and SNP typing using DNA of western flower thrips as a template; if the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as C:C, it was a homozygous sensitive individual; if the fluorescence signal data was typed as T:C, it was a heterozygous individual; the frequency of the resistance allele T in the tested population was counted, and the resistance level of the western flower thrips population to cypermethrin (pyrethroid) insecticide could be inferred.
[0014] (5) KASP primer combination Focc_T929C was used to perform KASP reaction and SNP typing using DNA of western flower thrips as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as A:A, it was a homozygous sensitive individual; if the fluorescence signal data was typed as T:A, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was counted, and the resistance level of the western flower thrips population to cypermethrin (pyrethroid) insecticide could be inferred.
[0015] (6) KASP primer combination Focc_L1014FGT was used to perform KASP reaction and SNP typing using DNA of western flower thrips as a template; if the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:T, it was a heterozygous individual; the frequency of the resistance allele T in the tested population was statistically analyzed, and the resistance level of the melon thrips population to cypermethrin (pyrethroid) insecticide could be inferred.
[0016] (7) KASP primer combination Musi_A355S was used to perform KASP reaction and SNP typing using the DNA of Thrips edulis as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:T, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was statistically analyzed to infer the resistance level of the Thrips edulis population to cyclopentadiene organochlorine insecticides.
[0017] (8) KASP primer combination Tapl_A355S was used to perform KASP reaction and SNP typing using the DNA of thrips as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:T, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was statistically analyzed to infer the resistance level of the thrips population to cyclopentadiene organochlorine insecticides.
[0018] (9) KASP primer combination Focc_A355S was used to perform KASP reaction and SNP typing using DNA of western flower thrips as a template; if the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:T, it was a heterozygous individual; the frequency of the resistance allele T in the tested population was counted, and the resistance level of the western flower thrips population to cyclopentadiene organochlorine insecticides could be inferred.
[0019] (10) KASP primer combination Musi_V386I was used to perform KASP reaction and SNP typing using DNA of Thrips edulis as a template. If the fluorescence signal data of the amplified product was typed as A:A, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele A in the tested population was statistically analyzed to infer the resistance level of the Thrips edulis population to bromofenac (diamide) insecticide.
[0020] (11) KASP primer combination Tapl_V386I was used to perform KASP reaction and SNP typing using the DNA of thrips as a template. If the fluorescence signal data of the amplified product was typed as A:A, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele A in the tested population was statistically analyzed to infer the resistance level of the thrips population to bromofenac (diamide) insecticide.
[0021] (12) KASP primer combination Focc_V386I was used to perform KASP reaction and SNP typing using DNA of western flower thrips as a template. If the fluorescence signal data of the amplified product was typed as A:A, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele A in the tested population was statistically analyzed to infer the resistance level of the western flower thrips population to bromofenac (diamide) insecticide.
[0022] (13) The KASP primer combination Fint_G275V was used to perform KASP reaction and SNP typing using the DNA of flower thrips as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:T, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was statistically analyzed to infer the resistance level of the melon thrips population to spinosad insecticides.
[0023] (14) KASP primer combination Focc_I1042M was used to perform KASP reaction and SNP typing using DNA of western flower thrips as a template. If the fluorescence signal data of the amplified product was typed as G:G, it was a homozygous resistant individual; if the fluorescence signal data was typed as C:C, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:C, it was a heterozygous individual. The frequency of the resistance allele G in the tested population was counted, and the resistance level of the western flower thrips population to triflumuron (benzoylurea) insecticide could be inferred.
[0024] The present invention has the beneficial effect of designing a KASP detection primer set for identifying resistant individuals, addressing the urgent need for rapid detection of pesticide resistance in thrips during production. This primer set enables rapid, accurate, low-cost, and high-throughput detection of pesticide resistance in thrips. This has broad application prospects in the rational and precise selection of pesticides. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 KASP genotype diagram for detecting drug-resistant mutations in thrips. DETAILED DESCRIPTION
[0026] The following describes in detail the method for detecting insecticide resistance of thrips using KASP primers designed in the present invention with reference to specific examples. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.
[0027] Example 1 KASP primer screening of mutation sites associated with drug resistance in thrips Genomic DNA sequences within 500 base pairs (bp) above and below the insecticide-resistance mutation site in thrips were extracted and used to design KASP primers using Primer3. KASP reactions and SNP typing were performed using thrips DNA as a template. Each KASP primer set was tested for clear typing and a success rate exceeding 90%. For each mutation site, a preferred combination was identified, as shown in Table 1.
[0028] Table 1 Preferred KASP primer sequence combinations obtained by screening
[0029]
[0030] Example 2 Application of KASP primers in detecting insecticide resistance of thrips
[0031] (1) Sample collection
[0032] Cucumber thrips samples were collected in 2022 from a plantation in Daxing District, Beijing (A) and a field population in a greenhouse at the Beijing Academy of Agricultural and Forestry Sciences (B). Both hosts were cucumbers. Western flower thrips samples were collected in 2017 from a plantation in Yanqing District, Beijing (A) and in 2022 from an organic farm in Huairou District, Beijing (B). Their hosts were cucumbers. Macrothrips leucophylla were collected in 2020 from farms in Wanning City, Hainan Province (A) and (B). Their hosts were cowpeas. Flower thrips were collected in 2021 from a plantation in Fangshan District, Beijing (A) and a plantation in Tongzhou District, Beijing (B). Live insects were collected, soaked in 100% alcohol, and stored at -20°C.
[0033] (2) DNA extraction
[0034] The genomic DNA was extracted from 46 individuals of populations A and B of Thrips citrinum, western flower thrips, Thrips macrocephala, and Thrips occidentalis using the DNA lysis and crude extraction method.
[0035] (3) KASP amplification, typing, and drug resistance prediction
[0036] The reagents used were 2× KASP master mixture (LGC). A 3 μL reaction system included 1.5 ng of dry powdered genomic DNA, 0.14 μL of primer mix (upstream primer concentration: 12 μmol / L, downstream primer concentration: 30 μmol / L), and 3 μL of 2× KASP master mixture.
[0037] The touchdown reaction procedure included: initial denaturation at 94°C for 15 min; followed by 10 cycles of denaturation at 94°C for 20 s and annealing at 61–55°C for 1 min, with a decrease of 0.6°C per cycle; and 40 cycles of denaturation at 94°C for 20 s and annealing at 55°C for 1 min.
[0038] Finally, check the typing status on the typing detector. Figure 1 The statistical genotype frequencies are shown in Table 2. The statistical allele frequencies are shown in Table 3.
[0039] Table 2 Genotype frequencies at resistance mutation sites in thrips populations A and B
[0040]
[0041] Table 3 Allele frequencies at the resistance mutation sites in thrips populations A and B
[0042]
Claims
1. A KASP primer combination for detecting drug-resistant mutations in thrips, characterized in that The kit includes KASP primers for detecting three mutation sites of citrullus thrips resistance to deltamethrin; KASP primers for detecting three mutation sites of citrullus thrips resistance to deltamethrin; KASP primers for detecting one mutation site of citrullus thrips resistance to cyclopentene organochlorine insecticides; KASP primers for detecting one mutation site of citrullus thrips resistance to cyclopentene organochlorine insecticides; KASP primers for detecting one mutation site of citrullus thrips resistance to cyclopentene organochlorine insecticides; KASP primers for detecting one mutation site of citrullus thrips resistance to cyclopentene organochlorine insecticides SP primers; KASP primers for detecting one mutation site of resistance of bean thrips to brofenoxamide; KASP primers for detecting one mutation site of resistance of melon thrips to brofenoxamide; KASP primers for detecting one mutation site of resistance of western flower thrips to brofenoxamide; KASP primers for detecting one mutation site of resistance of flower thrips to spinosad insecticides; KASP primers for detecting one mutation site of resistance of western flower thrips to triflumuron.
2. The KASP primer for detecting three mutation sites of melon thrips resistance to deltamethrin according to claim 1, characterized in that A primer combination comprising Tpal_M918TAG, Tpal_T929I and Tpal_L1014FGT; a KASP primer for detecting three mutation sites of resistance of western flower thrips to deltamethrin, characterized by comprising a primer combination of Focc_T929I, Focc_T929C and Focc_L1014FGT; a KASP primer for detecting one mutation site of resistance of bean thrips to cyclopentadiene organochlorine insecticides, characterized by comprising a primer combination of Musi_A355S; a KASP primer for detecting one mutation site of resistance of melon thrips to cyclopentadiene organochlorine insecticides, characterized by comprising a primer combination of Tpal_A355S; a KASP primer for detecting one mutation site of resistance of western flower thrips to cyclopentadiene organochlorine insecticides, characterized by comprising a primer combination of Tpal_A355S; a KASP primer for detecting one mutation site of resistance of melon thrips to cyclopentadiene organochlorine insecticides, characterized by comprising a primer combination of Tpal_A355S; a KASP primer for detecting one mutation site of resistance of western flower thrips to cyclopentadiene organochlorine insecticides KASP primers for detecting one mutation site of resistance to chlorine insecticides, characterized by including a primer combination of Focc_A355S; KASP primers for detecting one mutation site of resistance to brofenoxamide in bean thrips, characterized by including a primer combination of Musi_V386I; KASP primer combination for detecting one mutation site of resistance to brofenoxamide in melon thrips, characterized by including a primer combination of Tpal_V386I; KASP primers for detecting one mutation site of resistance to brofenoxamide in western flower thrips, characterized by including a primer combination of Focc_V386I; KASP primer combination for detecting one mutation site of resistance to brofenoxamide in flower thrips, characterized by including a primer combination of Fint_G275V; KASP primer combination for detecting one mutation site of resistance to bistrifluan in western flower thistle, characterized by including a primer combination of Focc_I1042M.
3. The primer combination for detecting Tpal_M918TAG according to claim 2 comprises two upstream primers and one downstream primer, whose sequences are shown in SEQ ID NOs.: 1-3, respectively; the primer combination for detecting Tpal_T929I comprises two upstream primers and one downstream primer, whose sequences are shown in SEQ ID NOs.: 4-6, respectively; the primer combination for detecting Tpal_L1014FGT comprises two upstream primers and one downstream primer, whose sequences are shown in SEQ ID NOs.: 7-9, respectively; the primer combination for detecting Focc_T929I comprises two upstream primers and one downstream primer, whose sequences are shown in SEQ ID NOs.: 10-12, respectively; the primer combination for detecting Focc_T929C comprises two upstream primers and one downstream primer, whose sequences are shown in SEQ ID NOs.: 13- 15; the sequences of the two upstream primers and one downstream primer comprised in the primer combination for detecting Focc_L1014FGT are shown in SEQ ID NOs.: 16-18, respectively; the sequences of the two upstream primers and one downstream primer comprised in the primer combination for detecting Musi_A355S are shown in SEQ ID NOs.: 19-21, respectively; the sequences of the two upstream primers and one downstream primer comprised in the primer combination for detecting Tpal_A355S are shown in SEQ ID NOs.: 22-24, respectively; the sequences of the two upstream primers and one downstream primer comprised in the primer combination for detecting Focc_A355S are shown in SEQ ID NOs.: 25-27, respectively; the sequences of the two upstream primers and one downstream primer comprised in the primer combination for detecting Musi_V386I are shown in SEQ ID NOs.: 28-30, respectively The sequences of the two upstream primers and one downstream primer comprised in the primer combination for detecting Tpal_V386I are shown in SEQ ID NOs.: 31-33, respectively; the sequences of the two upstream primers and one downstream primer comprised in the primer combination for detecting Focc_V386I are shown in SEQ ID NOs.: 34-36, respectively; the sequences of the two upstream primers and one downstream primer comprised in the primer combination for detecting Fint_G275V are shown in SEQ ID NOs.: 37-39, respectively; the sequences of the two upstream primers and one downstream primer comprised in the primer combination for detecting Focc_I1042M are shown in SEQ ID NOs.: 40-42, respectively.
4. The KASP primer combination of 14 resistance mutation sites according to claim 2, characterized in that: The sequences of the two upstream primers of each primer combination are respectively connected to different fluorescent modification tags.
5. A method for detecting drug-resistant mutations in thrips, characterized in that: The KASP primer combination according to claim 2 is used to detect whether there is a drug resistance-related mutation using the DNA of thrips as a template.
6. The method for detecting drug-resistant mutations in thrips according to claim 5, wherein: If the KASP primer combination Tpal_M918TAG is used, the sample with the SNP genotype of G:G is a homozygous individual for resistance, the genotype of A:A is a homozygous individual for sensitivity, and the genotype of G:A is a heterozygous individual; if the KASP primer combination Tpal_T929I is used, the sample with the SNP genotype of T:T is a homozygous individual for resistance, the genotype of C:C is a homozygous individual for sensitivity, and the genotype of C:T is a heterozygous individual; if the KASP primer combination Tpal_L1014FGT is used;The samples with SNP genotype of T:T are homozygous individuals for resistance, those with genotype of G:G are homozygous individuals for sensitivity, and those with genotype of G:T are heterozygous individuals; if the KASP primer combination Focc_T929I is used, the samples with SNP genotype of T:T are homozygous individuals for resistance, those with genotype of C:C are homozygous individuals for sensitivity, and those with genotype of T:C are heterozygous individuals; if the KASP primer combination Focc_T929C is used, the samples with SNP genotype of T:T are homozygous individuals for resistance, those with genotype of A:A are homozygous individuals for sensitivity, and those with genotype of A:T are heterozygous individuals; if the KASP primer combination Focc_L is used, the samples with SNP genotype of If the KASP primer combination Musi_A355S is used, the samples with the SNP genotype of T:T are homozygous individuals for resistance, the samples with the genotype of G:G are homozygous individuals for sensitivity, and the samples with the genotype of G:T are heterozygous individuals; if the KASP primer combination Musi_A355S is used, the samples with the SNP genotype of T:T are homozygous individuals for resistance, the samples with the genotype of G:G are homozygous individuals for sensitivity, and the samples with the genotype of T:G are heterozygous individuals; if the KASP primer combination Tapl_A355S is used, the samples with the SNP genotype of T:T are homozygous individuals for resistance, the samples with the genotype of G:G are homozygous individuals for sensitivity, and the samples with the genotype of T:G are heterozygous individuals; if the KASP primer combination If the KASP primer combination Focc_A355S is used, the samples with SNP genotype of T:T are homozygous individuals for resistance, those with genotype of G:G are homozygous individuals for sensitivity, and those with genotype of T:G are heterozygous individuals; if the KASP primer combination Musi_V386I is used, the samples with SNP genotype of A:A are homozygous individuals for resistance, those with genotype of G:G are homozygous individuals for sensitivity, and those with genotype of A:G are heterozygous individuals; if the KASP primer combination Tapl_V386I is used, the samples with SNP genotype of A:A are homozygous individuals for resistance, those with genotype of G:G are homozygous individuals for sensitivity, and those with genotype of A:G are heterozygous individuals; if the KASP primer combination Tapl_V386I is used, the samples with SNP genotype of A:A are homozygous individuals for resistance, those with genotype of G:G are homozygous individuals for sensitivity, and those with genotype of A:G are heterozygous individuals; If the SP primer combination Focc_V386I is used, samples with the SNP genotype of A:A are homozygous for resistance, those with the genotype of G:G are homozygous for sensitivity, and those with the genotype of A:G are heterozygous. If the KASP primer combination Fint_G275V is used, samples with the SNP genotype of T:T are homozygous for resistance, those with the genotype of G:G are homozygous for sensitivity, and those with the genotype of T:G are heterozygous. If the KASP primer combination Focc_I1042M is used, samples with the SNP genotype of G:G are homozygous for resistance, those with the genotype of C:C are homozygous for sensitivity, and those with the genotype of C:G are heterozygous.