CN115491425A - Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation detection method and application - Google Patents
Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation detection method and application Download PDFInfo
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Abstract
The invention discloses a rapid detection method for Acetylcholinesterase A201S and F290V mutation of spodoptera frugiperda based on a loop-mediated isothermal amplification (LAMP) technology, belonging to the field of pest drug resistance molecular detection and spodoptera frugiperda comprehensive control. The detection method comprises the steps of respectively designing 2 pairs of specific primers at the sites of the Spodoptera frugiperda acetylcholinesterase A201S and F290V, carrying out LAMP amplification, and judging whether the Spodoptera frugiperda acetylcholinesterase A201S and F290V mutant individuals exist according to the color of a reaction product. The method has the advantages of high stability, short time consumption, simple and convenient operation and high sensitivity, is suitable for molecular detection for rapidly detecting the drug resistance of spodoptera frugiperda to organophosphorus and carbamate insecticides in fields, and provides a decision basis for comprehensive control of spodoptera frugiperda.
Description
Technical Field
The invention belongs to the field of biological detection methods, and particularly relates to a rapid molecular detection method for Spodoptera frugiperda Acetylcholinesterase A201S and F290V mutant, which is used for high-sensitivity rapid molecular detection of Spodoptera frugiperda for target mutation of resistance of organophosphorus and carbamate insecticides acting on Acetylcholinesterase (AChE).
Background
Spodoptera frugiperda is an important agricultural pest which issues early warning to the world in 2018 United nations grain and agriculture organizations, and has the characteristics of high migration speed, strong reproductive capacity, heavy overeating, high prevention and control difficulty and the like. The insect finds invasion in Yunnan of China in 1 month in 2019, rapidly spreads to 26 provinces in China, colonizes in south China such as Yunnan, guangdong and Hainan, and is harmful to annual propagation. The Spodoptera frugiperda has a wide host range, can harm more than 350 crops such as rice, corn, wheat, sorghum, sugarcane, potato, peanut and the like, is mainly harmful to the corn after migrating into China, and poses serious threats to the corn production and grain safety of China. 9/15/2020, published by Ministry of agricultural rural "first class crop pest and disease records", and Spodoptera frugiperda causes huge agricultural loss and enrollment of pest and disease records.
At present, chemical control is still the fastest and effective means for controlling noctuid pests, and long-term excessive chemical control is relied on, so that the sensitivity of field noctuid populations to various insecticides is reduced, the problem of drug resistance is caused, and the difficulty of control is increased. The organophosphorus and carbamate insecticides are traditional agents for preventing and controlling spodoptera frugiperda, and the spodoptera frugiperda has high-level drug resistance to various organophosphorus and carbamate insecticides at present, and target genes of the organophosphorus and carbamate insecticidesace-1The a201S and F290V mutations were also detected. The early stage of gene sequencing shows that the Spodoptera frugiperda has 120 spodoptera frugiperda in 6 areas of Anhui provinceace-1The mutation frequencies of A201S and F290V of the gene are respectively as high as 50.0 percent and 81.7 percent, and Spodoptera frugiperda in field populationace-1The mutation frequency of the gene is obviously higher than that of a relatively sensitive strain in a laboratory, becauseThere is a need to further enhance Spodoptera frugiperda population in the fieldace-1Monitoring the mutation frequency of the gene.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method and is characterized in that 4 specific primers are designed for 6 regions of a target gene, constant-temperature amplification is carried out at 60 to 70 ℃ under the action of strand displacement DNA polymerase, and nucleic acid amplification of more than one billion times can be realized within 15 to 60 minutes. Upon DNA synthesis, pyrophosphate ions precipitated from deoxyribonucleic acid triphosphate substrates (dNTPs) reacted with magnesium ions in the reaction solution, and a large amount of magnesium pyrophosphate was precipitated and appeared white. WarmStart ® The LAMP color-changing premix comprises a visual pH indicator, a large amount of protons are generated in LAMP amplification reaction, and the pH value is caused to decrease, so that the color of the reaction solution is changed from pink to yellow. Therefore, the color change can be used as an index of the reaction, and whether amplification is performed or not can be identified only by visual observation without complicated electrophoresis and ultraviolet observation. As the loop-mediated isothermal amplification reaction does not need a special temperature-variable equipment PCR instrument and expensive reagents, the method has wide application prospect in field detection of gene types.
Disclosure of Invention
The invention aims to provide a rapid detection technology for Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation, and a convenient and accurate gene molecule detection method for Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation is established by solving four main technical links of specific primers of mutation sites, configuration of an LAMP reaction system, reaction temperature and time, result judgment standards and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation rapid molecular detection method, extracting Spodoptera frugiperda larva or imago genome DNA, and preparing a gDNA template; configuring an LAMP reaction system in a reaction vessel; and (5) performing constant-temperature incubation, and judging and detecting the gene mutation condition of the individual according to the color change.
In one aspect of the invention, the gDNA template in the LAMP reaction is prepared by extracting Genomic DNA of single Spodoptera frugiperda larvae or adults collected in the field according to the operation steps of a TIAnmp Genomic DNA Kit extraction Kit.
The LAMP reaction system of the present invention comprises 2
12.5 mu L of WarmStart LAMP premix reagent, 1.0 mu L of gDNA template, 2.5 mu L of mixture of inner primer FIP/BIP and outer primer F3/B3, 9.5 mu L of sterile water and the balance of 25 mu L of reaction system.
The concentration of the inner primer FIP/BIP is 1.0-2.0 mu mol.L -1 (ii) a The concentration of the outer primer F3/B3 of the invention is 0.2-1.0. Mu. Mol. L -1 。
The base sequence of an inner primer FIP-A201S for detecting the mutation of Spodoptera frugiperda acetylcholinesterase A201S is shown as SEQ ID No.1, the base sequence of an inner primer BIP-A201S is shown as SEQ ID No.2, the base sequence of an outer primer F3-A201S is shown as SEQ ID No.3, and the base sequence of an outer primer B3-A201S is shown as SEQ ID No. 4. By adding the above 2 pairs of primers, it was possible to achieve specific amplification of homo-and heterozygous mutants for Ala (GCG) 201Ser (TCG), but not Ala (GCG) 201 non-mutant.
The base sequence of an inner primer FIP-F290V for detecting the mutation of Spodoptera frugiperda acetylcholinesterase F290V is shown as SEQ ID No.5, the base sequence of an inner primer BIP-F290V is shown as SEQ ID No.6, the base sequence of an outer primer F3-F290V is shown as SEQ ID No.7, and the base sequence of an outer primer B3-F290V is shown as SEQ ID No. 8. By adding the above 2 pairs of primers, it was possible to achieve specific amplification of Phe (TTT) 290Val (GTT) homozygous and heterozygous mutants, whereas it was not possible to amplify Phe (TTT) 290 non-mutant.
The constant-temperature incubation is characterized in that a special PCR instrument is not needed, the constant temperature is kept in a water bath kettle, a metal bath or a vacuum flask at 60-70 ℃, and the constant-temperature incubation time is 30-90 min.
The method for judging and detecting the gene mutation condition of the individual according to the color change is characterized in that a sample containing A201S or F290V mutation generates a large amount of protons in LAMP amplification reaction, so that the color of a reaction solution is changed into yellow; the samples without the A201S and F290V mutations were not amplified efficiently and the reaction solution was still pink in color.
In one aspect of the invention, the invention also provides a spodoptera frugiperda acetylcholinesterase A201S and F290V mutation detection kit, wherein the mutation detection kit can be provided with the specific combination of an inner primer and an outer primer; preferably, the kit also comprises a spodoptera frugiperda gene extraction reagent; LAMP reaction reagent.
In one aspect of the invention, the invention also provides application of a Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation rapid detection technology, and the rapid detection technology is used for controlling Spodoptera frugiperda.
In one aspect of the invention, the invention also discloses an application of the Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation detection kit, and the kit is used for LAMP rapid detection of Spodoptera frugiperda acetylcholinesterase A201S and F290V so as to control Spodoptera frugiperda.
Advantageous effects
Compared with the prior art of in vivo bioassay, conventional molecular biology sequencing and the like, the method has the following beneficial effects:
the LAMP-based nucleic acid amplification method is adopted, the target fragments can still be amplified in the template with extremely low amount under the condition that the amplification template has 10 copies or less, the amplification product is more than one billion times of the original amount of the template, and the amplification product is 100 to 1000 times higher than that of the traditional PCR detection technology, and the sensitivity is high.
The inner primers and the outer primers for detecting the Spodoptera frugiperda acetylcholinesterase A201S and F290V mutations, which are screened by the invention, are respectively 2 pairs, LAMP amplification can be carried out only under the condition that the 2 pairs of primers are completely matched with 6 sections on an acetylcholinesterase gene, LAMP amplification can not be carried out only if one base cannot be matched, and the detection accuracy is high.
The optimized constant-temperature incubation temperature and time are 68 ℃ and 80 min, while the ordinary PCR reaction needs special temperature changing equipment and takes 90 min, and the electrophoresis detection and sequencing confirmation need 2 to 3 days to complete. Therefore, the invention has the advantages of short time consumption and simple operation.
The Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation inner primer and outer primer designed by the invention can perform LAMP amplification no matter whether the homozygous mutation or the heterozygous mutation occurs when mutation occurs in Ala (GCG) 201Ser (TCG) and Phe (TTT) 290Val (GTT), so that the reaction solution is changed from pink to yellow, while Ala (GCG) 201 and Phe (TTT) 290 non-mutants cannot be amplified, and the reaction solution is still pink. The on-site high-flux rapid detection is realized without any special instrument and equipment, the detection result is easy to identify, and the method is suitable for detection of plant protection personnel at the basic level.
Drawings
FIG. 1 shows the LAMP primer design position of Spodoptera frugiperda acetylcholinesterase A201S mutation;
FIG. 2 shows the LAMP primer design position of Spodoptera frugiperda acetylcholinesterase F290V mutation;
FIG. 3 shows LAMP detection results of Spodoptera frugiperda acetylcholinesterase A201S mutation, wherein A is a color change chart; b is an electrophoresis detection image; c is a gene sequencing peak map;
FIG. 4 shows LAMP detection results of Spodoptera frugiperda acetylcholinesterase F290V mutation, wherein A is a color change chart; b is an electrophoresis detection image; c is a gene sequencing peak map;
FIG. 5 is a LAMP detection graph of mutations in Acetylcholinesterase A201S of Spodoptera frugiperda in the field, wherein A is a color change graph; b is an electrophoresis detection image; c is a gene sequencing peak map;
FIG. 6 LAMP detection map of Spodoptera frugiperda acetylcholinesterase F290V mutation in field, where A is a color change map; b is an electrophoresis detection image; c is a gene sequencing peak map.
Detailed Description
The following detailed description of the embodiments of the invention is provided in connection with the examples, but it should be understood that the scope of the invention is not limited to the specific embodiments.
Example 1: specificity and accuracy verification test of LAMP reaction
1. Experimental materials
Insect DNA extraction Kit TIANAmp Genomic DNA Kit was purchased from Tiangen, plasmid extraction Kit FastPeure Plasmid Mini Kit, DNA polymerase, RNase and nucleic acid scavenger were purchased from Nanjing Triazan, warmStart Colorimetric LAMP (M1800S) was purchased from NEB, and RNase-free double-distilled sterile water was purchased from Bio Inc.
2. Experimental procedure
2.1 Extraction of genomic DNA of Spodoptera frugiperda
(1) Collecting field spodoptera frugiperda larvae or adults in different areas of Anhui province, and respectively collecting the larvae or adults in different sterilized centrifugal tubes.
(2) And pouring liquid nitrogen into the mixture, and grinding the cleaned spodoptera frugiperda larvae or imagoes into powder.
(3) The powder was centrifuged to the bottom of the tube, 200. Mu.L of buffer GA was added, and shaken to complete suspension.
(4) 20 μ L of protease K was added thereto, and the mixture was left at 56 ℃ for 3 hours. Mix the samples by inversion 2-3 times per hour.
(5) Adding 200 μ L buffer GB, mixing thoroughly, standing at 70 deg.C for 10 min, and clearing the solution.
(6) Add 200. Mu.L of absolute ethanol and shake well for 15 s, at which time a flocculent precipitate may appear.
(7) Adding the solutions in the previous step into an adsorption column CB3 (placing the adsorption column into a collecting pipe), centrifuging at 12000 rpm for 30 s, pouring the waste liquid, and placing the adsorption column CB3 back into the collecting pipe.
(8) Add 500. Mu.L buffer GD to adsorption column CB3, centrifuge at 12000 rpm for 30 s, pour off the waste, and place adsorption column CB3 back into the collection tube.
(9) 600. Mu.L of the rinsing solution PW was added to the adsorption column CB3, and the mixture was centrifuged at 12000 rpm for 30 seconds, and the waste liquid was discarded, and the adsorption column CB3 was returned to the collection tube.
(10) And (3) putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12000 rpm for 2 min, pouring waste liquid, and placing the adsorption column CB3 at room temperature for a plurality of minutes to completely dry the residual rinsing liquid in the adsorption material.
(11) Transferring the adsorption column CB3 into a clean centrifugal tube, suspending and dripping 50 mu L of elution buffer TE into the central part of the adsorption membrane, standing for 5 min at room temperature, centrifuging at 12000 rpm for 2 min, and collecting the spodoptera frugiperda genome DNA solution into the centrifugal tube for later use.
2.2 Cloning and identification of Spodoptera frugiperda acetylcholinesterase gene
Downloading genomic data of spodoptera frugiperda in a national gene bank (https:// db. Cngb. Org/cnsa /), obtaining a genomic sequence of spodoptera frugiperda acetylcholinesterase (> 3339514-3341240 position of chr 6) by homology search, and designing upstream and downstream primers for cloning sites comprising the codes A201 and F290 according to the sequence. The sequence of the upstream primer is as follows: tcagaggcattcacgttaac; the sequence of the downstream primer is as follows: TGTTTGCAACTTCTAGGGAA. The primers were synthesized by Chuzhou general biology, and were purified by RPC. The PCR reaction for amplifying the genomic sequence of Spodoptera frugiperda AChE is as follows: 2. 12.5 mu L of x Phanta Flash Master Mix, 1.0 mu L of spodoptera frugiperda genome template, 1.0 mu L of each of upstream and downstream primers (10 mu M), and 9.5 mu L of sterile water; the reaction program was pre-denatured at 95 ℃ for 3 min, and the following 3 process cycles 35 times: 95. denaturation at 15 s, annealing at 60 deg.C for 15 s, extension at 72 deg.C for 2 min, and complete extension at 72 deg.C for 5 min. And (3) carrying out agarose gel electrophoresis on the PCR amplification product, detecting whether the size of a target band accords with experimental expectation in an ultraviolet gel imaging system, cutting the target band into gel, and sending the gel to Chuzhou general biology company for sequencing and identification.
2.2 Design and screening of LAMP primers
According to the verified genome sequence of Spodoptera frugiperda AChE, designing LAMP primers by using an online design website http:// primer explorer.jp/e/index.html according to an LAMP design primer principle, designing LAMP primers by using a primer explorer V5 program, and performing primer specificity screening according to two mutation sites needing to be detected of Ala (GCG) 201Ser (TCG) and Phe (TTT) 290Val (GTT), wherein 6 sites for combining the LAMP primers of A201S and F290V are respectively shown as an attached figure 1 and an attached figure 2, and specific primer sequences are shown as the following table:
a201S primer set:
F290V primer set:
2.3 Configuration and incubation of LAMP reaction system
Firstly, diluting all LAMP primers to 10 mu M, wherein an A201S primer group is mixed according to the proportion of 16 mu L of FIP primer, 16 mu L of BIP primer, 4 mu L of F3 primer and 4 mu L of B3 primer; the F290V primer set was mixed at a ratio of 16. Mu.L FIP primer, 16. Mu.L BIP primer, 2. Mu.L F3 primer and 2. Mu.L B3 primer. The following components were added to a 200 μ L centrifuge tube using the WarmStart Colorimetric LAMP (M1800S) kit from NEB: a25.0-. Mu.L LAMP reaction system was prepared from WarmStart Colorimetric LAMP 2 × Master Mix 12.5. Mu.L, spodoptera frugiperda genomic template 1.0. Mu.L, LAMP primer mixture (10 ×) 2.5. Mu.L, and sterile water 9.0. Mu.L.
Sealing the prepared LAMP reaction system by using a sealing film, and then putting the sealed LAMP reaction system into a water bath kettle to heat at 68 ℃ for 60-90 min to observe color change. After the color change, 3. Mu.L of the sample was taken and checked by 2% agarose gel electrophoresis.
3. Results of the experiment
Through screening and experimental verification, the binding positions of the LAMP primer group mutated by Spodoptera frugiperda acetylcholinesterase A201S and the template are shown in the attached figure 1.
6 binding positions of the LAMP primer group mutated by Spodoptera frugiperda acetylcholinesterase F290V and a template are shown in the attached figure 2 through screening and experimental verification.
Through the color change of the LAMP reaction system (A in the attached figure 3), the detection of an electrophoretic band (B in the attached figure 3) and the gene sequencing verification (C in the attached figure 3), the LAMP amplification can be carried out by confirming that the 201 site GCG → TCG homozygous mutation (lane 1) and the GCG → G/TCG heterozygous mutation (lane 4) can both change the reaction solution from pink to yellow, while the 201 site GCG non-mutant (lanes 2 and 5) and the clear water control (lanes 3 and 6) can not be amplified, and the reaction solution is still pink.
Through the color change of the LAMP reaction system (A in figure 4), the electrophoresis band detection (B in figure 4) and the gene sequencing verification (C in figure 4), the LAMP amplification can be carried out on the confirmed 290 sites TTT → GTT homozygous mutant (lane 1) and TTT → G/TTT heterozygous mutation (lane 4), so that the reaction solution is changed from pink to yellow, the 290 sites TTT non-mutant (lanes 2 and 5) and the clear water control (lanes 3 and 6) cannot be amplified, and the reaction solution is still pink.
Example 2: field application for rapidly detecting mutations of Acetylcholinesterase A201S and F290V of Spodoptera frugiperda
1. Experimental Material
The experimental materials used in this section were the same as in example 1.
2. Experimental procedure
2.1 Extraction and preparation of field spodoptera frugiperda genome DNA
The extraction and preparation steps of the Spodoptera frugiperda genome DNA in the part of the field are the same as those of the Spodoptera frugiperda genome DNA in the example 1.
2.2 Sequencing detection of Acetylcholinesterase A201S and F290V mutation of Spodoptera frugiperda in field
The sequencing detection steps of the Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation in the part of fields are the same as the cloning and identification of the Spodoptera frugiperda acetylcholinesterase gene in the example 1.
2.3 LAMP detection of Acetylcholinesterase A201S and F290V mutation of Spodoptera frugiperda in field
The LAMP detection steps of the Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation in the field are the same as the configuration and incubation of the LAMP reaction system in the example 1.
3. Results of the experiment
The LAMP amplification can be carried out by confirming that 201 sites GCG → TCG homozygous mutation (lane 1) and GCG → G/TCG heterozygous mutation (lanes 2 and 3) can be changed from pink to yellow through the color change of the LAMP reaction system (A in figure 5), electrophoretic band detection (B in figure 5) and gene sequencing verification (C in figure 5); while the GCG non-mutant at position 201 (lanes 4 and 5), the TTT → GTT homozygous mutant at position 290 (lanes 6 and 7), the TTT non-mutant at position 290 (lanes 8 and 9) and the clear water control (lane 10) were not discolored. The DNA sequencing result is consistent with the LAMP detection result, and the rapid detection method for Spodoptera frugiperda acetylcholinesterase A201S mutation, which is established by the invention, has high accuracy, and the accuracy rate is up to 100%.
The LAMP amplification can be carried out by confirming that 290 sites TTT → GTT homozygous mutant ( lanes 1, 2 and 3) and TTT → G/TTT heterozygous mutation ( lanes 4, 5 and 6) can be amplified through the color change of the LAMP reaction system (A in figure 6), electrophoretic band detection (B in figure 6) and gene sequencing verification (C in figure 6), so that the reaction solution is changed from pink to yellow; while the TTT non-mutant at position 290 (lanes 7 and 8), GCG → TCG homozygous mutant at position 201 (lanes 9 and 10), GCG non-mutant at position 201 (lanes 11 and 12) and the clear water control (lane 13) were not discolored. The DNA sequencing result is consistent with the LAMP detection result, and the rapid detection method for Spodoptera frugiperda acetylcholinesterase F290V mutation, which is established by the invention, has high accuracy, and the accuracy rate is up to 100%.
Various other modifications and changes may be made by those skilled in the art based on the above teachings and concepts, and all such modifications and changes are intended to fall within the scope of the appended claims.
Claims (10)
1. A primer combination for detecting Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation based on loop-mediated isothermal amplification (LAMP) technology is characterized in that,
the primer combination comprises an inner primer FIP/BIP and an outer primer F3/B3, wherein the sequences of the inner primer FIP/BIP are respectively as follows:
FIP-A201S：TCGATAGAGGTGACAATAAATGCAAAACATTATTCGGTGAGTCGT（SEQ ID NO.1）
BIP-A201S： AACTTGTTCTCTCAGGCGATAATGTCAAAATGCTTTCTTCTCTTGA（SEQ ID NO.2）
FIP-F290V：GCTCATCTAGGAACGAACCATCTAGGAACTCTTGGTATTTGTGAAG（SEQ ID NO.5）
BIP-F290V：TACCCGCTCGATCATTAGCAGTAGTAACCTTCCTCCGTATT（SEQ ID NO.6）
the sequences of the outer primers F3/B3 are respectively as follows:
F3-A201S：TACTTCGGAGGAAACCCG（SEQ ID NO.3）
B3-A201S：TTCTGCTAATCGGATGCC（SEQ ID NO.4）
F3-F290V：GATGAACTAGTCAATAATGAATGG（SEQ ID NO.7）
B3-F290V：CCTTGGGGAATAACTCAGT（SEQ ID NO.8）。
2. a method for detecting Spodoptera frugiperda acetylcholinesterase A201S and F290V mutation based on loop-mediated isothermal amplification (LAMP) technology, which is characterized by comprising the following steps:
(1) Extracting genome DNA of spodoptera frugiperda larvae or adults, and preparing a gDNA template;
(2) An LAMP reaction system is established in a reaction vessel, and comprises LAMP reaction liquid, specific primers and a gDNA template, and sterile water is supplemented;
(3) Placing the LAMP reaction system into a constant-temperature container for constant-temperature incubation reaction;
(4) And (4) judging a result: after the reaction is finished, the sample of which the reaction solution does not change color is a non-mutant sample, and the sample of which the reaction solution changes color is a mutant sample;
wherein the specific primer comprises an inner primer FIP/BIP and an outer primer F3/B3, and the sequences of the inner primer FIP/BIP are respectively as follows:
FIP-A201S：TCGATAGAGGTGACAATAAATGCAAAACATTATTCGGTGAGTCGT（SEQ ID NO.1）
BIP-A201S： AACTTGTTCTCTCAGGCGATAATGTCAAAATGCTTTCTTCTCTTGA（SEQ ID NO.2）
FIP-F290V：GCTCATCTAGGAACGAACCATCTAGGAACTCTTGGTATTTGTGAAG（SEQ ID NO.5）
BIP-F290V：TACCCGCTCGATCATTAGCAGTAGTAACCTTCCTCCGTATT（SEQ ID NO.6）
the sequences of the outer primers F3/B3 are respectively as follows:
F3-A201S：TACTTCGGAGGAAACCCG（SEQ ID NO.3）
B3-A201S：TTCTGCTAATCGGATGCC（SEQ ID NO.4）
F3-F290V：GATGAACTAGTCAATAATGAATGG（SEQ ID NO.7）
B3-F290V：CCTTGGGGAATAACTCAGT（SEQ ID NO.8）。
3. the spodoptera frugiperda acetylcholinesterase A201S and F290V mutation detection method according to claim 2, wherein in step (2), the LAMP reaction solution comprises 2
WarmStart LAMP premix.
4. The method for detecting Spodoptera frugiperda acetylcholinesterase A201S and F290V mutants according to claim 3, wherein the LAMP reaction system comprises LAMP reaction liquid including 2
12.5 mu L of WarmStart LAMP premix reagent, 1.0 mu L of gDNA template, 2.5 mu L of mixture of inner primer FIP/BIP and outer primer F3/B3 and the balance of sterile water to 25 mu L of reaction system.
5. The method for detecting mutations in acetylcholinesterase A201S and F290V of Spodoptera frugiperda according to claim 4, wherein the concentration of said inner primer FIP/BIP is 1.0-2.0. Mu. Mol.L -1 (ii) a The concentration of the outer primer F3/B3 is 0.2-1.0 mu mol.L -1 。
6. The method for detecting mutations of acetylcholinesterase A201S and F290V of spodoptera frugiperda according to claim 2, wherein in step (3), the incubation reaction is carried out at a constant temperature, and the heating temperature is 60-70 ℃ and the heating time is 20-60 min.
7. The method for detecting mutations A201S and F290V in Spodoptera frugiperda acetylcholinesterase according to claim 2, wherein in step (4), the color of the reaction solution containing the mutation A201S or F290V changes to yellow, and the color of the reaction solution not containing the mutation A201S or F290V remains pink.
8. A spodoptera frugiperda acetylcholinesterase A201S and F290V mutation detection kit, characterized in that the kit contains the primer combination of claim 1.
9. A spodoptera frugiperda acetylcholinesterase A201S and F290V mutation detection kit, characterized in that the kit comprises a spodoptera frugiperda larva or imago genome DNA extraction reagent, LAMP reaction liquid, and the primer combination of claim 1.
10. The use of the detection kit of any one of claims 9 to 10 for detecting spodoptera frugiperda control, characterized in that the detection kit is used for detecting the mutations of a201S and F290V in spodoptera frugiperda acetylcholinesterase.
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