LAMP primer set, kit and application thereof for detecting wheat straw fly

Abstract

The present invention belongs to the technical field of straw fly identification, and specifically relates to a LAMP primer set, a kit and an application thereof for detecting straw flies. The LAMP primer set includes an outer primer F3 shown in SEQ ID NO.1, an outer primer B3 shown in SEQ ID NO.2, an inner primer FIP shown in SEQ ID NO.3 and an inner primer BIP shown in SEQ ID NO.4. The present invention also provides a kit for detecting straw flies, an application of the LAMP primer set and the kit in identifying and / or detecting straw flies, and a method for detecting straw flies using the LAMP primer set. The method has the advantages of strong specificity, high sensitivity, simple operation process, low detection cost, etc., solves the problem of distinguishing straw flies, and lays a foundation for quarantine inspection, epidemic monitoring and prevention and control of straw flies in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of straw fly identification, and particularly relates to a LAMP primer set and a kit for detecting straw flies and applications thereof. Background Art

[0002] Elachiptera decipiens (Loew) is a member of the family Chloropida, superfamily Carnoidea, order Diptera, class Acalyptrata. The family can be divided into four subfamilies: Chloropinae, Rhodesiellinae, Siphonellopsinae, and Oscinellinae. It is estimated that there are about 5,000 to 10,000 members of the family Chloropinae worldwide, and 200 genera and 3,000 species have been recorded.

[0003] The wheat straw fly, El. decipiens, originated in North America. It not only harms wheat, but also forage grass, causing a reduction in forage production, which seriously affects the development of animal husbandry. The wheat straw fly, El. decipiens, also causes "dead seedlings" in the wheat seedling stage. Therefore, it is difficult to distinguish different types of wheat straw flies based solely on the symptoms of the damage.

[0004] The identification of straw flies mainly relies on the morphological characteristics of adults. The morphological identification method has extremely high requirements on the integrity of the specimens and the professional level of the identifier; the larvae and pupae are currently unable to be identified based on morphological characteristics. In addition, when using sexual trapping technology for epidemic monitoring, many non-target species are often trapped on the insect trap plates, and the captured insect bodies are often damaged and difficult to identify morphologically. Molecular identification using conventional PCR amplification technology takes a long time and cannot be done quickly. Therefore, it is very necessary to develop fast, accurate, simple and reliable species identification technology products. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a LAMP primer set, a kit and an application thereof for detecting wheat straw flies in view of the deficiencies of the above-mentioned prior art. The LAMP primer set has the advantages of strong specificity, high sensitivity, simple operation process and low detection cost, and solves the problem that wheat straw flies cannot be quickly identified during quarantine and field investigation, laying a foundation for quarantine inspection, epidemic monitoring and prevention and control of wheat straw flies in the future.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A LAMP primer set for detecting straw flies, comprising an outer primer F3 shown in SEQ ID NO.1, an outer primer B3 shown in SEQ ID NO.2, an inner primer FIP shown in SEQ ID NO.3 and an inner primer BIP shown in SEQ ID NO.4.

[0008] The invention also provides a kit for detecting wheat straw flies, comprising the LAMP primer set.

[0009] Furthermore, the kit also includes: a LAMP reaction mixture and Bca DNA polymerase.

[0010] Furthermore, the LAMP reaction mixture includes: 10×Isothermal Amplification Buffer II, MgSO4 solution, dNTP Mix, betaine, and sterile distilled water.

[0011] The present invention also provides the use of the LAMP primer set or the kit in identifying and / or detecting straw flies.

[0012] The present invention also provides a method for identifying straw flies, comprising the following steps:

[0013] 1) extracting DNA from the insect to be tested to obtain genomic DNA to be tested;

[0014] 2) establishing a LAMP reaction system using the genomic DNA to be detected obtained in step 1), and performing LAMP amplification on the genomic DNA to be detected using the LAMP primer set to obtain a LAMP product;

[0015] 3) adding hydroxynaphthofine blue dye to the LAMP product, and according to the color development result, if the solution turns sky blue after the reaction is completed, the insect body to be tested in step 1) is a straw fly, and if the solution is purple, the insect body to be tested in step 1) is not a straw fly; or the LAMP product is subjected to 2% agarose gel electrophoresis to check the amplification result, and if a ladder-shaped DNA band appears, the insect body to be tested in step 1) is a straw fly.

[0016] Furthermore, the reaction system of LAMP amplification in step 2) is: 2.5 μL of 10×IsothermalAmplification BufferII, 1.5 μL of 100 mM MgSO4 solution, 3.5 μL of 10 mM dNTP Mix, 1 μL of 40 μM inner primer FIP, 1 μL of 40 μM inner primer BIP, 0.5 μL of 10 μM outer primer F3, 0.5 μL of 10 μM outer primer B3, 5 μL of 5 M betaine, 1 μL of 8000 U / mL Bca DNA polymerase, 1 μL of genomic DNA to be detected, and the volume is filled to 25 μL with sterile distilled water.

[0017] Furthermore, the reaction procedure of LAMP amplification in step 2) is: isothermal reaction at 64° C. for 60 min.

[0018] Furthermore, in step 3), the concentration of the hydroxynaphthofine blue dye is 250 μM, and the dosage is 1 μL.

[0019] Compared with the prior art, the present invention has the following technical advantages and positive effects:

[0020] 1. High specificity: The present invention designs 4 specific primers for 6 regions of the target gene based on the whole genome of the COI sequence of the wheat straw fly Elachiptera decipiens (Loew). If any region in the 6 regions does not match the primer, the amplification effect cannot be achieved within 60 minutes, so the specificity is extremely high;

[0021] 2. High sensitivity: The detection sensitivity of the present invention to wheat straw fly DNA can reach 10 at the DNA level. -3 ng / μL, which has high sensitivity;

[0022] 3. Efficient and rapid detection: The detection method of the present invention can obtain the detection result within 50 minutes, while the previous PCR or qPCR detection requires 1.5 to 2 hours to obtain the detection result. The method of the present invention greatly shortens the operation time and is convenient and rapid;

[0023] 4. Strong applicability: The LMAP amplification reaction of the present invention is amplified under a constant temperature condition, and does not require an expensive PCR amplification instrument, but only a simple water bath or heating equipment. It is combined with the toothpick method to extract DNA, which is convenient for grassroots promotion and use;

[0024] 5. Low cost: This method does not require expensive and sophisticated test instruments and equipment such as PCR instruments. It only requires a constant temperature water bath or other constant temperature equipment. In addition, conventional reagents are used in the experimental process, which are low in price.

[0025] 6. The test results are intuitive and can be judged by naked eyes: the positive reaction in the amplification product of the present invention is sky blue, and the negative reaction is purple, and the test results can be judged only by naked eyes.

[0026] In summary, the LAMP detection method has the advantages of strong specificity, high sensitivity, simple operation process, low detection cost, and visualized detection results. It can provide technical support for the rapid detection of wheat straw flies in the field and customs quarantine departments, and can provide a scientific basis for early prediction of the pest, timely formulation of pest control strategies and effective prevention and control. It has important guiding significance, broad application prospects and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the primer set designed for LAMP detection based on the COI gene of the straw fly Elachiptera decipiens (Loew).

[0028] Figure 2 Specificity determination of 4 sets of wheat straw fly LAMP primer sets, M: 2000bp DNA Marker, N: enzyme-free sterile water; ED: wheat straw fly, ET: tuberculous straw fly, HMGY: black rye straw fly, HF: Hessian gall midge, repeated three times; a: primer set 1, b: primer set 2, c: primer set 3, d: primer set 4.

[0029] Figure 3 Optimization of reaction temperature for LAMP detection of wheat straw flies, reaction temperature 53.0℃~65℃, reaction time: 60min; M: 2000bp DNA Marker, N: enzyme-free sterile water. 1: 53.0℃, 2: 54.1℃, 3: 55.8℃, 4: 57.0℃, 5: 57.7℃, 6: 57.8℃, 7: 58.9℃, 8: 60.2℃, 9: 60.5℃, 10: 62.0℃, 11: 62.7℃, 12: 64.2℃, 13: 65℃. a: colorimetric results, b: electrophoresis results; sky blue indicates positive, purple indicates negative.

[0030] Figure 4 To optimize the reaction time of LAMP detection of wheat straw flies, the reaction temperature is 54.1℃, the reaction time is 30min-60min; M: 2000bp DNA Marker, N: enzyme-free sterile water; 1: 30min, 2: 40min, 3: 50min, 4: 60min, repeated three times; a: colorimetric result, b: electrophoresis result; sky blue indicates positive, purple indicates negative.

[0031] Figure 5For sensitive detection of wheat straw fly genomic DNA; M: 2000bp DNA Marker, N: enzyme-free sterile water; 1: DNA concentration is 100ng / μL, 2: 10ng / μL, 3: 1ng / μL, 4: 10 -1 ng / μL, 5:10 -2 ng / μL, 6:10 -3 ng / μL, 7:10 - 4 ng / μL; a: colorimetric result, b: electrophoresis result; sky blue indicates positive, and purple indicates negative.

[0032] Figure 6 In order to simulate the field environment, the toothpick method was used to extract the DNA of the insects to be tested for LAMP detection; M: 2000bp DNA Marker, N: enzyme-free sterile water; a: colorimetric results of Xinjiang wheat stalk fly samples, 1-3: adults, 4-6: larvae, 7-10: pupae; b: colorimetric results of Shaanxi samples, 1-2: gray ground fly D. platura, 3-4: pea leafminer: Ch. Horticola, 5-6: barley tooth-horned yellow leafminer Ce. denticornis, 7-8: thoracic fly Se. thoracica, 9-10: Sc. atomaria; c: electrophoresis results of Xinjiang samples; d: electrophoresis results of Shaanxi samples; sky blue indicates positive and purple indicates negative. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0034] The invention provides a LAMP primer set for detecting straw flies, comprising a FIP primer, a BIP primer, an F3 primer and a B3 primer; the nucleotide sequence of the FIP primer is shown in SEQ ID NO.1; the nucleotide sequence of the BIP primer is shown in SEQ ID NO.2; the nucleotide sequence of the F3 primer is shown in SEQ ID NO.3; and the nucleotide sequence of the B3 primer is shown in SEQ ID NO.4. The present invention determines the COI sequence of the straw fly Elachiptera decipiens (Loew) and other closely related species, compares and analyzes the COI sequence, and uses the online LAMP primer design software Primer software Explorer V5 (https: / / primerexplorer.jp / lampv5 / index.html; Eiken Chemical Co., Japan) to design 4 sets of straw fly LAMP primer sets, each set of primers includes upstream and downstream outer primers F3 and B3 and upstream and downstream inner primers FIP and BIP, wherein the upstream inner primer FIP is composed of two primer fragments F2 and F1c, and the downstream inner primer BIP is composed of two primer fragments B2 and B1c, such as Figure 1 shown.

[0035] The invention also provides a kit for detecting straw flies, comprising the LAMP primer set, a LAMP reaction mixture and Bca DNA polymerase; the LAMP reaction mixture comprises: 10×Isothermal Amplification Buffer II, MgSO4 solution, dNTP Mix, betaine and sterile distilled water.

[0036] The present invention also provides the use of the primer set or the kit in identifying and / or detecting straw flies.

[0037] The present invention also provides a method for identifying straw flies, comprising the following steps:

[0038] 1) extracting DNA from the insect to be tested to obtain genomic DNA to be tested;

[0039] 2) establishing a LAMP reaction system using the genomic DNA to be detected obtained in step 1), and performing LAMP amplification on the genomic DNA to be detected using the LAMP primer set to obtain a LAMP product;

[0040] 3) adding hydroxynaphthofine blue dye to the LAMP product, and according to the color development result, if the solution turns sky blue after the reaction is completed, the insect body to be tested in step 1) is a straw fly, and if the solution is purple, the insect body to be tested in step 1) is not a straw fly; or the LAMP product is subjected to 2% agarose gel electrophoresis to check the amplification result, and if a ladder-shaped DNA band appears, the insect body to be tested in step 1) is a straw fly.

[0041] When the present invention is used to detect the insect body to be tested, the kit method is used to extract the genomic DNA of the insect body to be tested, and the specific method is as follows:

[0042] Extraction using DNA kit

[0043] (1) Collect the sample and place it in a sterile 1.5 mL centrifuge tube; after freezing with liquid nitrogen, add small steel beads and grind it thoroughly with a tissue grinder, then add 200 μL of GA and shake until it is thoroughly suspended;

[0044] (2) Add 20 μL of Proteinase K solution, mix well, and add in a water bath at 65°C for 1 h, inverting several times to ensure thorough mixing;

[0045] (3) Add 200 μL of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 min. The solution should become clear and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0046] (4) Add 200 μL of anhydrous ethanol, vortex and mix thoroughly for 15 seconds, and briefly centrifuge;

[0047] (5) Add the solution obtained in the previous step to the adsorption column, place the adsorption column in a collection tube, centrifuge at 12000r for 30s, discard the waste liquid, and place the adsorption column back into the collection tube;

[0048] (6) Add 500 μL of buffer GD to the adsorption column, centrifuge under the same conditions, discard the waste liquid, and return the adsorption column to the collection tube;

[0049] (7) Add 600 μL of rinse solution PW to the adsorption column, centrifuge under the same conditions, discard the waste liquid and put the adsorption column back;

[0050] (8) Repeat the previous step;

[0051] (9) Centrifuge at 12000 r / min for 2 min, discard the waste liquid, and leave the adsorption column to dry at room temperature for several minutes;

[0052] (10) Transfer the adsorption column to a clean centrifuge tube, add 50-200 μL of elution solution TE to the middle of the adsorption film, let it stand at room temperature for 2-5 min, and centrifuge it at 12000 r / min to obtain the DNA solution. Detect the DNA concentration using a UV spectrophotometer and dilute it to 100 ng / μL for later use.

[0053] When the present invention is used to detect whether the insect body to be tested in the field is a wheat straw fly, a toothpick method is used to extract DNA. The specific process is as follows: the insect body to be tested is rinsed with sterile water and then placed in a 1.5 mL centrifuge tube, a sterile toothpick is used to pierce the insect body to make its body fluid ooze out, 30 μL of TE buffer is added, and then the insect body is placed in a 95° water bath for 2 minutes, and 1.0 μL of the supernatant is taken as a LAMP template for amplification.

[0054] In the present invention, the LAMP amplification system preferably contains per 25 μL: 0.5 μL of 10 μM F3 primer and B3 primer, 1 μL of 40 μM FIP primer and BIP primer, 1 μL of 20 μM LF probe and LB loop primer, 12.5 μL of LAMP reaction mixture, 1 μL of 8000U / mL Bca DNA polymerase and 1 μL of genomic DNA to be detected, and sterile water is added to 25 μL. In the present invention, the conditions of the LAMP amplification are preferably: temperature of 65°C and time of 30 min.

[0055] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1: Design of LAMP primer set for detecting straw flies

[0057] The mitochondrial cytochrome oxidase (COI) genes of Elachiptera decipiens (Loew) and other closely related species were determined, and the COI genes were compared and analyzed. Four sets of LAMP primer sets for Elachiptera decipiens (Loew) were designed using the online LAMP primer design software Primersoftware Explorer V5 (https: / / primerexplorer.jp / lampv5 / index.html; Eiken Chemical Co., Japan). Each set of primers included an upstream outer primer F3, a downstream outer primer B3, an upstream inner primer FIP, and a downstream inner primer BIP. The sequences of the four sets of primers are detailed in Table 1, where primer set 1 is a specific primer for LAMP for Elachiptera decipiens (Loew). The specific sites are shown in Table 1. Figure 1 All four primer sets were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0058] Table 1 Primer and probe sequences for LAMP-LFD detection of wheat straw flies

[0059]

[0060]

[0061] Example 2: Specificity verification of the LAMP-LFD detection method for wheat straw flies

[0062] 1. Extraction of genomic DNA from insects to be tested

[0063] The kit method was used to extract the genomic DNA of the insects to be tested (Table 2). The specific method was as follows: collect the samples and place them in a sterile 1.5 mL centrifuge tube; after freezing with liquid nitrogen, add small steel beads and grind them thoroughly with a tissue grinder, then add 200 μL of GA and shake until completely suspended; add 20 μL of Proteinase K solution, mix well, add in a water bath at 65℃ for 1h, invert several times during the period to mix thoroughly; add 200μL buffer GB, invert thoroughly to mix, place at 70℃ for 10min, the solution should be clear, and centrifuge briefly to remove water droplets on the inner wall of the tube cap; add 200μL anhydrous ethanol, oscillate and mix thoroughly for 15s, and centrifuge briefly; add the solution obtained in the previous step to the adsorption column, put the adsorption column into the collection tube, centrifuge at 12000r for 30s, pour out the waste liquid, and put the adsorption column back into the collection tube; add 500μL buffer GD to the adsorption column, centrifuge under the same conditions and pour out the waste liquid, and put the adsorption column back into the collection tube; add 600μL rinse solution PW to the adsorption column, centrifuge under the same conditions, pour out the waste liquid and put the adsorption column back; repeat the previous step; centrifuge at 12000r for 2min, pour out the waste liquid, and let the adsorption column dry at room temperature for several minutes; transfer the adsorption column to a clean centrifuge tube, suspend 50 -200μL elution solution TE, place at room temperature for 2-5min, centrifuge at 12000r for 2min to obtain DNA solution, detect DNA concentration with UV spectrophotometer and dilute to 10ng / μL for later use.

[0064] Table 2. Samples of tested species used for specificity verification of LAMP-LFD of wheat straw flies

[0065]

[0066] 2. Determination of LAMP Assay Specificity

[0067] The established LAMP detection system was used to amplify the genomic DNA of the insect body to be tested using 4 sets of primers to evaluate its specificity, with the genomic DNA of the wheat straw fly as the positive control and sterile distilled water as the negative control. The amplified products were detected by 2% agarose gel electrophoresis.

[0068] 3. Specificity Verification Results

[0069] Extract the genomic DNA of the insects to be tested in Table 2, and use the established LAMP for specific detection. The specific detection results of the four sets of LAMP primers are as follows: Figure 2 As shown in (a-d), among the four primer sets, only the first primer set showed a positive reaction to the wheat straw fly ( Figure 2 ), and the other tested insects were all negative, indicating that the designed first group of wheat straw fly LAMP primers can distinguish wheat straw flies from other pests, have stable specificity, and can be used for rapid and reliable detection and identification of wheat straw flies. Therefore, the first group of primers was selected as the specific primers for the wheat straw fly LAMP system.

[0070] Example 3: Establishment of LAMP detection method for wheat straw flies

[0071] Optimization of LAMP amplification conditions: The genomic DNA of the test insects of wheat straw flies was extracted using conventional nucleic acid extraction methods or commercial kits (such as the plant genomic DNA extraction kit of Tiangen Biotechnology Co., Ltd.) as a template for LAMP amplification. The LAMP reaction system was prepared in a sterile 200 μL Eppendorf tube. The various reagents and final concentrations in the 25 μL reaction system were: 0.5 μL of 10 μM F3 primer and B3 primer, 1 μL of 40 μM FIP primer and BIP primer, 2.5 μL of 10×IsothermalAmplification BufferII, 1.5 μL of 100 mM MgSO4 solution, 3.5 μL of 10 mM dNTP Mix, 5 μL of 5 M betaine, 1 μL of 8000 U / mL Bca DNA polymerase and 1 μL of genomic DNA to be tested, and sterile distilled water was added to 25 μL.

[0072] In order to obtain a reaction system with the best amplification efficiency and detection specificity, the reaction temperature and amplification time of LAMP amplification were optimized. First, the reaction temperature of LAMP amplification was optimized. Too high or too low amplification temperature was not conducive to LAMP reaction. The prepared 25μL reaction system was placed at different temperatures (53.0℃, 54.1℃, 55.8℃, 57.0℃, 57.7℃, 57.8℃, 58.9℃, 60.2℃, 60.5℃, 62.0℃, 62.7℃, 64.2℃, 65℃) for LAMP amplification reaction for 60min, and the effect of different temperatures on LAMP reaction was evaluated to screen out the optimal reaction temperature. After determining the optimal reaction temperature, the LAMP reaction system was placed at the optimal reaction temperature (54.1℃) for 30min, 40min, 50min and 60min respectively to optimize the optimal reaction time. The optimal reaction conditions (temperature, time) were finally optimized from multiple repeated experiments.

[0073] LAMP amplification product detection: 2% agarose gel electrophoresis and colorimetry are used to detect the LAMP amplification results. Agarose gel electrophoresis: Take 3.0 μL of LAMP amplification product for 2% agarose gel electrophoresis. If a ladder-like band appears in the gel, it means the test result is positive. If there is no ladder-like band in the gel, it means the test result is negative. Colorimetric method: Add hydroxynaphthofine blue dye to the LAMP product. According to the color development results, if the solution turns sky blue after the reaction, the insect to be tested is a straw fly. If the solution is purple, the insect to be tested is not a straw fly.

[0074] Establishment of LAMP detection method: The optimization results of LAMP reaction temperature showed that positive reaction could occur under the conditions of 54.1℃-65.0℃. The brightness of electrophoresis band and test strip detection line was the strongest at 54.1℃, indicating that the optimal reaction temperature of LAMP was 54.1℃( Figure 3 ).

[0075] At the optimal reaction temperature, the results of different reaction times were analyzed and the optimization results of wheat straw fly LAMP showed that ( Figure 4 ), a positive reaction was observed at 60 min of LAMP amplification reaction, and both DNA electrophoresis bands and colorimetry were obvious, so 60 min was selected as the optimal reaction time.

[0076] According to the optimization results of reaction temperature and time, the final straw fly LAMP-LFD detection system was determined to be 25 μL, including 12.5 μL of LAMP reaction mixture, 0.5 μL of 10 μM outer primers (F3 and B3), 1 μL of 40 μM inner primers (FIP and BIP), 1 μL of 8000 U / mL Bca DNA polymerase and 1 μL of genomic DNA to be detected. Sterile distilled water was added to 25 μL, and the reaction conditions were 54.1°C for 60 min.

[0077] Example 4: Sensitivity determination of the LAMP assay

[0078] 1. Preparation of Genomic DNA at Different Concentrations

[0079] The genomic DNA of the wheat straw fly was diluted with sterile distilled water to prepare a series of concentrations of 10 times the order of magnitude for later use.

[0080] 2. Sensitivity determination of LAMP detection method and observation of results

[0081] Using different concentrations of straw fly genomic DNA as template, LAMP amplification was performed using primers F3 / B3 and primers FIP / BIP. The LAMP detection reaction system was 25 μL, including 0.5 μL each of 10 μM primers F3 and B3, 1.0 μL each of 40 μM primers FIP and BIP, 12.5 μL of LAMP reaction mixture (10×Isothermal Amplification BufferII 2.5 μL, 100 mM MgSO4 solution 1.5 μL, 10 mM dNTP Mix 3.5 μL, 5 M betaine 5 μL), 1.0 μL of 8000 U / mL Bca polymerase, 1.0 μL of DNA templates of different concentrations, and made up to 25 μL with sterile distilled water; LAMP reaction conditions: incubation at 54°C for 60 min; the amplified products were detected by 2% agarose gel electrophoresis and colorimetry, respectively.

[0082] 3. LAMP amplification sensitivity test results

[0083] Using different concentrations of genomic DNA of wheat straw flies as templates, the established LAMP detection system was used for sensitivity detection. 2% agarose gel electrophoresis and colorimetry were used to detect the amplified products.

[0084] The results of the genomic DNA concentration sensitivity test showed that ( Figure 5 ), 10ng, 1ng, 10 -1 ng, 10 -2 ng, 10 -3 ng / μL concentration of wheat straw fly genomic DNA was positive, while the other concentrations and negative control were negative, indicating that the detection sensitivity of the established LAMP for wheat straw fly was 10 -3 ng / μL.

[0085] Example 5: Combined application of toothpick method for crude DNA extraction and LAMP rapid detection technology in simulated field environment

[0086] 1. Preparation of the insects to be tested: Rinse the insects to be tested with sterile water and place them in a 1.5 mL centrifuge tube containing 30 μL TE buffer.

[0087] 2. Toothpick method for crude extraction of DNA from insects: Use a sterile toothpick to fully crush the insects soaked in TE buffer to mix their body fluids in the TE buffer. Then place the centrifuge tube in a 95°C water bath for 2 minutes, and take the supernatant as a DNA template for LAMP amplification detection.

[0088] 3. LAMP test results of crude DNA of test insects: 1 μL of the supernatant as the test insect DNA template was used as a template for LAMP amplification test. The LAMP amplification system was as in Example 2, with the genomic DNA of the test insects of the straw fly as the positive control and sterile distilled water as the negative control. The results of the combined application of the toothpick method and LAMP showed that all the test insects of the straw fly were positive, and the sterile water control was negative ( Figure 6 ), indicating that the LAMP detection system for wheat straw flies established in this study can provide a simple, rapid and sensitive on-site detection for wheat straw flies.

[0089] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0090] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A LAMP primer set for detecting wheat straw flies, characterized in that: The LAMP primer set includes an outer primer F3 as shown in SEQ ID NO.1, an outer primer B3 as shown in SEQ ID NO.2, an inner primer FIP as shown in SEQ ID NO.3 and an inner primer BIP as shown in SEQ ID NO.

4.

2. A kit for detecting wheat straw flies, characterized in that: The invention comprises the LAMP primer set according to claim 1.

3. The kit according to claim 2, characterized in that Also includes: LAMP reaction mix and Bca DNA polymerase.

4. The kit according to claim 3, characterized in that The LAMP reaction mixture includes: 10×Isothermal Amplification Buffer II, MgSO4 solution, dNTP Mix, betaine, and sterile distilled water.

5. Use of the LAMP primer set according to claim 1 or the kit according to any one of claims 2 to 4 in identifying and / or detecting straw flies.

6. A method for identifying straw flies, characterized in that: The following steps are involved: 1) Extract the DNA of the insect to be tested to obtain the genomic DNA to be tested; 2) establishing a LAMP reaction system using the genomic DNA to be detected obtained in step 1), and performing LAMP amplification on the genomic DNA to be detected using the LAMP primer set of claim 1 to obtain a LAMP product; 3) adding hydroxynaphthofine blue dye to the LAMP product, and according to the color development result, if the solution turns sky blue after the reaction is completed, the insect body to be tested in step 1) is a straw fly, and if the solution is purple, the insect body to be tested in step 1) is not a straw fly; or the LAMP product is subjected to 2% agarose gel electrophoresis to check the amplification result, and if a ladder-shaped DNA band appears, the insect body to be tested in step 1) is a straw fly.

7. The method for identifying straw flies according to claim 6, characterized in that: The reaction system of LAMP amplification in step 2) is: 2.5 μL of 10×Isothermal Amplification Buffer II, 1.5 μL of 100 mM MgSO4 solution, 3.5 μL of 10 mM dNTP Mix, 1 μL of 40 μM inner primer FIP, 1 μL of 40 μM inner primer BIP, 0.5 μL of 10 μM outer primer F3, 0.5 μL of 10 μM outer primer B3, 5 μL of 5 M betaine, 1 μL of 8000 U / mL Bca DNA polymerase, 1 μL of genomic DNA to be detected, and the volume is filled to 25 μL with sterile distilled water.

8. The method for identifying straw flies according to claim 7, characterized in that: The reaction procedure of LAMP amplification in step 2) is: isothermal reaction at 64°C for 60 minutes.

9. The method for identifying straw flies according to claim 8, characterized in that: The concentration of the hydroxynaphthofine blue dye in step 3) is 250 μM, and the dosage is 1 μL.

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