Primer set, identification method and identification kit for codling moth based on LAMP method

Abstract

The invention discloses a primer set, an identification method and an identification kit for codling moth based on the LAMP method, and belongs to the technical field of agricultural pest identification. The primer set for codling moth identification based on the LAMP method of the invention is designed according to the nucleic acid sequence shown in SEQ ID NO: 1; the primer set is used to perform a LAMP reaction with the whole genome DNA or part of the tissue of the insect to be tested as a template; after the reaction is completed, whether there is an amplification product; if there is amplification, the insect to be tested is codling moth. The invention uses a rapid molecular detection plus a visualization method to identify codling moth, and obtains the detection result within 1 hour. The visualization detection method without opening the cover can effectively avoid aerosol pollution, can accurately develop color, and the color development result is visible to the naked eye, does not require expensive instruments and equipment, and is suitable for rapid identification of codling moth on site and at the grassroots level.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural pest identification, and in particular relates to a primer set, an identification method and an identification kit for codling moth based on a LAMP method. Background Art

[0002] The codling moth (Cydia pomonella) belongs to the family Tortricidae, order Lepidoptera. With its strong reproductive capacity, it has occurred in more than 70 countries around the world. It is a very serious pest in apple-producing areas and is a devastating pest to the fruit industry. The pest has a wide host range and mainly harms more than 30 kinds of fruit trees, such as apples, pears, and hawthorns. It mainly causes damage in the larval stage, with a fruit infestation rate of more than 50%, and in the most serious cases, it can reach 70% to 100%, seriously affecting the quality of the fruit. Some of the fruit cannot even be eaten, thus losing its due economic value. It can be seen that monitoring and preventing codling moths and eliminating the damage caused by codling moths are the top priorities for achieving high-quality and high-efficiency fruits.

[0003] Because the codling moth is small in size and begins to cause damage in the larval stage, it often occurs in secret, causing losses to the environment and economy, thus seriously affecting fruit production and international trade. It is listed as an important imported plant quarantine pest by many countries and regions.

[0004] For a long time, the identification of codling moth has been mainly based on its morphological characteristics. Due to different living environments, the morphological characteristics of codling moth will change accordingly, which makes identification difficult. Moreover, during the quarantine of fruits and vegetables, codling moth larvae are usually detected. It is difficult to accurately identify codling moth larvae through traditional morphological methods, which brings difficulties to the quarantine and prevention of codling moth.

[0005] At present, although there are some specific primer identification technologies for codling moth. For example, the Chinese patent application with application number 201811568937.3 discloses codling moth-specific SS-COII primers and their applications. The designed primers can specifically detect codling moth, and there is no amplification product in its closely related species, the genus Borer. However, using PCR technology, the entire process needs to be completed within 5 hours, which is time-consuming and cumbersome. The methods for molecular detection of codling moth are mainly based on polymerase chain reaction (PCR) technology or real-time fluorescence quantitative (Real-time PCR), all of which have the disadvantages of long test cycle, cumbersome operation, limited test materials, and high technical requirements for experimental instruments and test personnel. Therefore, it is difficult to meet the detection needs of grassroots laboratories. Summary of the invention

[0006] In view of the problems existing in the prior art, the object of the present invention is to provide a method and application for detecting codling moth based on visualized LAMP technology.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A primer set for identifying codling moth based on a LAMP method is designed according to the nucleic acid sequence shown in SEQ ID NO:1.

[0009] In a specific embodiment, the primer set consists of an F3 primer with a nucleotide sequence of SEQ ID NO:2, a B3 primer with a nucleotide sequence of SEQ ID NO:3, a FIP primer with a nucleotide sequence of SEQ ID NO:4, and a BIP primer with a nucleotide sequence of SEQ ID NO:5.

[0010] The application of the above-mentioned LAMP method-based primer set for identifying codling moth in the identification of codling moth.

[0011] A method for identifying codling moth, using the above primer set, taking the whole genome DNA of the insect to be tested or part of the tissue of the insect to be tested as a template, to carry out LAMP reaction; after the reaction is completed, detecting whether there is an amplification product; if there is amplification, the insect to be tested is codling moth.

[0012] The partial tissue of the insect to be tested may be the leg, thorax or abdomen tissue of an adult, or may be partial abdominal segment tissue of a larva.

[0013] In a specific embodiment, the conditions of the LAMP reaction are: 63° C. for 45 min, and inactivation at 80° C. for 10 min.

[0014] In a specific embodiment, a nucleic acid dye is added to the system after the LAMP reaction to detect whether an amplification product exists; the nucleic acid dye is calcein or SYBR Green I, and in a specific embodiment, it is SYBRGreen I.

[0015] In a specific embodiment, the nucleic acid dye is added in the following manner: after the LAMP reaction system is prepared, a movable diaphragm that does not contact the reaction system is set at the upper end of the reaction tube, and the nucleic acid dye is dripped onto the diaphragm, and then the lid is closed for reaction. After the reaction is completed, the nucleic acid dye on the diaphragm is mixed into the system after the reaction by centrifugation, and the color development result is observed to determine whether there is an amplification product. The diaphragm is preferably tin foil or aluminum foil. The above operation realizes the operation of color development without opening the lid, avoiding the problem of cross contamination during the operation.

[0016] A portable kit for identifying codling moth based on visualized LAMP technology, comprising a LAMP primer set, wherein the LAMP primer set consists of an F3 primer with a nucleotide sequence of SEQ ID NO: 2, a B3 primer with a nucleotide sequence of SEQ ID NO: 3, a FIP primer with a nucleotide sequence of SEQ ID NO: 4, and a BIP primer with a nucleotide sequence of SEQ ID NO: 5.

[0017] In a specific embodiment, it also includes nucleic acid dye, Bst DNA large fragment polymerase, 10×ThermoPol buffer, 100mmol / L MgSO4, 10mmol / L dNTPs mixed solution, 5mol / L Betaine, a movable diaphragm and a constant temperature tool; wherein the constant temperature tool can be selected as a travel cup (brand is Tanxunjia, model TXJ-U1, an electric water cup with adjustable temperature).

[0018] Advantages of the technical solution of the present invention:

[0019] The present invention solves the problem that it is difficult to identify the larvae of the codling moth. The rapid molecular detection plus visualization method can obtain the test results within 1 hour. The visual detection method without opening the cover can effectively avoid aerosol pollution and can accurately display colors. No expensive instruments and equipment are required. A simple constant temperature device can achieve the reaction. The result detection is also very simple. The green fluorescence can be directly observed by the naked eye. Unlike the ordinary PCR method, gel electrophoresis is required to observe the results. It is a method suitable for on-site and grassroots rapid detection.

[0020] The method and primer set provided by the present invention can accurately detect codling moth among species of the genus Cichorius, and have high sensitivity, and can be used for visual rapid testing of test materials in biological quarantine. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the color development effect of SYBR Green I;

[0022] Figure 2 The primer set for codling moth has specificity in detecting codling moth populations in different regions;

[0023] Figure 3 Test results of some tissue samples of codling moth;

[0024] Figure 4 The primer set for codling moth has detection specificity for different types of pests;

[0025] Figure 5 This is the sensitivity test result of the primer set for codling moth. DETAILED DESCRIPTION

[0026] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0027] The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0028] Example 1

[0029] A primer set for detecting codling moth is designed based on a 641 bp fragment (SEQ ID NO: 1) on the mitochondrial COI gene of codling moth.

[0030] Wherein, the sequence of SEQ ID NO:1 is as follows:

[0031]

[0032] Primers were designed online using PrimerExplorer V5 (URL link: http: / / primerexplorer.jp / lampv5e / index.html) to design primers specific for codling moth. More than 300 sets of potential primers were designed, and 7 sets of potential primers were finally selected by excluding primers with high repetitiveness, as shown in Table 1. After repeated verification, the five sets of primers named 01, 34, 50, 655, and 874 were non-specific and did not amplify. Only a few positive primers of 331 were amplified, and the results were non-specific. Finally, 03 primers were determined to be specific primers, which had 100% successful amplification of codling moth and did not amplify other similar pests tested.

[0033] Table 1 Primers for LAMP detection of Cydia pomonella

[0034]

[0035]

[0036] Example 2

[0037] A method for identifying codling moth, the steps are as follows:

[0038] a) taking whole body or partial tissue samples of the insect to be tested and extracting genomic DNA as a DNA template;

[0039] b) performing a LAMP reaction using specific amplification primers: FIP / BIP and F3 / B3, wherein the F3 nucleic acid sequence is shown in SEQ ID NO:2, the B3 nucleic acid sequence is shown in SEQ ID NO:3, the FIP nucleic acid sequence is shown in SEQ ID NO:4, and the BIP nucleic acid sequence is shown in SEQ ID NO:5.

[0040] LAMP reaction system:

[0041] 20μL reaction system: 2μL of 10×ThermoPol buffer, 0.8μL of MgSO4 (100mmol / L), 2.8μL of dNTPs mixed solution (10mmol / L), 4μL each of FIP / BIP (10μm / L), 0.5μL each of F3 / B3 (10μm / L), 1.0μL of Betaine (5mol / L), 0.8μL of Bst DNA large fragment polymerase (8U / μL), 1μL of DNA template, add water to 20μL.

[0042] Reaction conditions: 63℃ for 45min, inactivation at 80℃ for 10min.

[0043] c) Visual detection of reaction products:

[0044] After the LAMP reaction system is prepared, a movable diaphragm that does not contact the reaction system is set at the upper end of the reaction tube, and the nucleic acid dye is dripped on the diaphragm, and then the lid is closed for reaction. After the reaction is completed, the nucleic acid dye on the diaphragm is mixed into the system after the reaction by centrifugation, and the color development result is observed to determine whether there is an amplification product. If the color changes, it means that amplification has occurred, and the insect to be tested is codling moth; if the color of the reaction system does not change, that is, no amplification has occurred, then the insect to be tested is not codling moth; for example, when the nucleic acid dye used is SYBR Green1, the color development effect is as follows Figure 1 As shown, from left to right: 1-3 are codling moth, 4-5 are pear borer, 6 is a blank control, Figure 1 It can be seen that SYBR Green1 can clearly distinguish between positive (green) and negative results (brown). Therefore, the test results can be successfully observed by using the lid-free method in the color development step.

[0045] Example 3

[0046] Specificity of primer sets for detecting codling moth populations in different regions.

[0047] Since there is a certain variation in the genetic information of individuals of codling moth from different regions, if the primers can be successfully amplified on the target individuals, it means that the primers are effective. The codling moth trapped by trap monitoring was used as the detection object (Table 2). All the tested insects were verified by adult morphological identification and DNA barcoding.

[0048] Table 2 Tested insects

[0049]

[0050] The LAMP reaction detection steps are as follows:

[0051] a) Take whole body or partial tissue samples of the insect to be tested, and use the TIANamp Genomic DNA Kit to extract genomic DNA according to the instructions of the kit.

[0052] b) performing a LAMP reaction using specific amplification primers: FIP / BIP and F3 / B3, wherein the F3 nucleic acid sequence is shown in SEQ ID NO:2, the B3 nucleic acid sequence is shown in SEQ ID NO:3, the FIP nucleic acid sequence is shown in SEQ ID NO:4, and the BIP nucleic acid sequence is shown in SEQ ID NO:5.

[0053] LAMP reaction system:

[0054] 20μL reaction system: 10×ThermoPol buffer 2μL, MgSO4 (100mmol / L) 0.8μL, dNTPs mixed solution (10mmol / L) 2.8μL, FIP / BIP (10μm / L) 4μL each, F3 / B3 (10um / L) 0.5μL each, Betaine (5mol / L) 1.0μL, Bst DNA large fragment polymerase (8U / uL) 0.8μL, DNA template 1μL, add water to 20μL.

[0055] Put it in a constant temperature tool, 63℃ for 45 minutes, and inactivate at 80℃ for 10 minutes. The constant temperature tool can be a travel cup (brand is Tanxunjia, model is TXJ-U1, an electric water cup with adjustable temperature).

[0056] c) Visual detection of reaction products:

[0057] After the reaction system is prepared, place a piece of tin foil on the upper layer of the inner wall of the reaction tube, so that it is suspended in the air, drop 1μL of nucleic acid dye (SYBR Green I) on the tin foil, cover the lid, and centrifuge with a palm-sized centrifuge after the reaction is completed to allow the nucleic acid dye to mix into the system. If the color turns green, it means that amplification has occurred. The color of the negative control system is brown.

[0058] The test results are as follows:

[0059] Since the genetic structure of codling moths in different regions varies to a greater or lesser extent, if the primers cannot amplify these sites, the test effect will be affected. In this embodiment, amplification verification was performed on codling moths collected from different regions. Through verification of 12 codling moths from 4 regions, the amplification rate was 100% (e.g. Figure 2 ).

[0060] Example 4

[0061] Insect abdominal tissue (about one-tenth of the size of abdominal tissue) was selected. No DNA extraction was required. Part of the tissue was directly placed in the reaction system (2 μL of 10×ThermoPol buffer, 0.8 μL of MgSO4 (100 mmol / L), 2.8 μL of dNTP mixture (10 mmol / L), 4 μL each of FIP / BIP (10 μm / L), 0.5 μL each of F3 / B3 (10 μm / L), 1.0 μL of Betaine (5 mol / L), 0.8 μL of Bst DNA large fragment polymerase (8 U / μL), and water was added to 20 μL). The temperature was kept constant in a travel cup (brand name Tanxunjia, model number TXJ-U1, an electric water cup with adjustable temperature), 63°C for 45 min, inactivated at 80°C for 10 min, and the final reactant was detected. The results are as follows: Figure 3 Specifically, 1-3 codling moth (abdominal tissue), 4-5 pear borer (abdominal tissue), 6-7 blank control. From the results, the method of the present invention can be used without DNA extraction, and a suitable amount of insect tissue can be directly taken for detection.

[0062] Example 5

[0063] Different types of pests were tested to test the specificity of LAMP primers. Insects other than codling moth are shown in Table 3. All samples were sequenced and aligned to ensure correct species identification. Deionized water was used as a blank control instead of DNA template.

[0064] Table 3 Insects other than codling moth

[0065]

[0066] The LAMP reaction was performed using specific amplification primers: FIP / BIP and F3 / B3, wherein the F3 nucleic acid sequence is shown in SEQ ID NO: 2, the B3 nucleic acid sequence is shown in SEQ ID NO: 3, the FIP nucleic acid sequence is shown in SEQ ID NO: 4, and the BIP nucleic acid sequence is shown in SEQ ID NO: 5. The specific detection method is the same as in Example 2.

[0067] Test results such as Figure 4 As shown, Figure 4 The fluorescence color development results are as follows: from left to right, they are numbered 1-3, codling moth, 4, pear borer, 5, beet armyworm, 6, Spodoptera litura, and 7, blank control. From the above test results, it can be seen that the primer set of the present invention has high specificity.

[0068] Example 6

[0069] The specific amplification primers SEQ ID NO: 2-SEQ ID NO: 5 were used to perform LAMP reaction on different initial concentrations of the codling moth DNA template, and the sensitivity of the primer set was determined by detecting the amplification results of different concentrations.

[0070] The concentration of the mother solution of the codling moth DNA template was 231.35 ng / μL. 1 μL of the mother solution was added with 9 μL ddH2O for 10-fold gradient dilution. The dilution gradient was 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Deionized water was used instead of DNA template as blank control. The specific detection method was the same as in Example 2.

[0071] The test results were observed using an ultra-micro UV spectrophotometer (N60). Figure 5 As shown, Figure 5 The eight samples from left to right are original concentration, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , blank control. Figure 5 It can be seen that at 231.35×10 -3 ng / μL template concentration, the fluorescence intensity can still be distinguished from the blank control. Therefore, the minimum detection concentration of the method of the present invention can reach 231.35×10 -3 ng / μL, corresponding to the 4th tube in the figure.

[0072] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention. Sequence Listing <110> Qingdao Agricultural University <120> Primer set, identification method and identification kit for codling moth based on LAMP method <160> 29 <170> SIPOSequenceListing 1.0 <210> 1 <211> 641 <212> DNA <213> Codling moth (Cydia pomonella) <400> 1 taaagatatt ggaacattat attttatttt tggtatttga gccggaatag taggaacttc 60 tctaagatta cttattcgag cagaattagg aaatccagga tctttaattg gtgatgatca 120 aatttataat actattgtaa ctgctcatgc ttttattata atttttttta tagtaatacc 180 tattataatt ggtggatttg gtaattgatt agtaccacta atattaggag ctcctgatat 240 agcttttcct cgaataaata atataagatt ttgattatta cccccatcta ttatactttt 300 aatttcaagc agaatcgttg aaaatggagc aggaacagga tgaacagtgt accccccact 360 ttcttctaat attgcccata gaggaagatc tgttgattta gctatttttt ctctgcattt 420 agcaggtatt tcttctattt taggagctgt taattttatt acaactatta ttaatatacg 480 acctaataat atatcattag atcaaatacc attatttatt tgagctgtag gaattacagc 540 tcttttactt cttttatcat taccagtatt agcaggtgct attactatgc ttcttacaga 600 tcgaaatctt aatacatcat tttttgaccc tgctggtgga g 641 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 cctgatatag cttttcctcg a 21 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <400> 3 cctaaaatag aagaaatacc tgctaa 26 <210> 4 <211> 47 <212> DNA <213> Artificial Sequence <400> 4 tccattttca acgattctgc ttgaaagatt ttgattatta cccccat 47 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <400> 5 aacaggatga acagtgtacc cccagagaaa aaatagctaa atcaacag 48 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 ctattataat tggtggattt ggt 23 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 tcttcctcta tgggcaata 19 <210> 8 <211> 49 <212> DNA <213> Artificial Sequence <400> 8 agatgggggt aataatcaaa atcttacact aatattagga gctcctgat 49 <210> 9 <211> 44 <212> DNA <213> Artificial Sequence <400> 9 acttttaatt tcaagcagaa tcgttgaaga aagtgggggg taca 44 <210> 10 <211> twenty three <212> DNA <213> Artificial Sequence <400> 10 ctattataat tggtggattt ggt 23 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <400> 11 tcttcctcta tgggcaata 19 <210> 12 <211> 48 <212> DNA <213> Artificial Sequence <400> 12 agatgggggt aataatcaaa atcttcacta atattaggag ctcctgat 48 <210> 13 <211> 44 <212> DNA <213> Artificial Sequence <400> 13 acttttaatt tcaagcagaa tcgttggaag aaagtggggg gtac 44 <210> 14 <211> twenty three <212> DNA <213> Artificial Sequence <400> 14 ttggtaattg attagtacca cta 23 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <400> 15 acctgctaaa tgcagagaa 19 <210> 16 <211> 51 <212> DNA <213> Artificial Sequence <400> 16 gcttgaaatt aaaagtataa tagatggggg agctcctgat atagcttttc c 51 <210> 17 <211> 44 <212> DNA <213> Artificial Sequence <400> 17 agaatcgttg aaaatggagc agggatcttc ctctatgggc aata 44 <210> 18 <211> twenty three <212> DNA <213> Artificial Sequence <400> 18 ctattataat tggtggattt ggt 23 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <400> 19 cctgctaaat gcagagaa 18 <210> 20 <211> 47 <212> DNA <213> Artificial Sequence <400> 20 agatgggggt aataatcaaa atcttcacta atattaggag ctcctga 47 <210> twenty one <211> 43 <212> DNA <213> Artificial Sequence <400> twenty one aagcagaatc gttgaaaatg gagatcttcc tctatgggca ata 43 <210> twenty two <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty two ggatttggta attgattagt acc 23 <210> twenty three <211> 20 <212> DNA <213> Artificial Sequence <400> twenty three tacctgctaa atgcagagaa 20 <210> twenty four <211> 47 <212> DNA <213> Artificial Sequence <400> twenty four agatgggggt aataatcaaa atcttactaa tattaggagc tcctgat 47 <210> 25 <211> 43 <212> DNA <213> Artificial Sequence <400> 25 aagcagaatc gttgaaaatg gagatcttcc tctatgggca ata 43 <210> 26 <211> twenty three <212> DNA <213> Artificial Sequence <400> 26 ctattataat tggtggattt ggt 23 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <400> 27 tcttcctcta tgggcaata 19 <210> 28 <211> 49 <212> DNA <213> Artificial Sequence <400> 28 agatgggggt aataatcaaa atcttacact aatattagga gctcctgat 49 <210> 29 <211> 44 <212> DNA <213> Artificial Sequence <400> 29 acttttaatt tcaagcagaa tcgttgagaa agtggggggt acac 44

Claims

1. Application of a primer set for identifying codling moth based on the LAMP method in identifying codling moth, characterized in that: The LAMP-based primer set for identifying codling moth consists of an F3 primer with a nucleotide sequence of SEQ ID NO: 2, a B3 primer with a nucleotide sequence of SEQ ID NO: 3, a FIP primer with a nucleotide sequence of SEQ ID NO: 4, and a BIP primer with a nucleotide sequence of SEQ ID NO:

5.

2. A method for identifying codling moth, characterized in that: A primer set for identifying codling moth based on the LAMP method is used, and the whole genome DNA of the insect to be tested or part of the tissue of the insect to be tested is used as a template to perform a LAMP reaction; after the reaction is completed, whether there is an amplification product is detected; if there is amplification, the insect to be tested is codling moth; The conditions of the LAMP reaction are: 63°C for 45 min, inactivation at 80°C for 10 min; By adding nucleic acid dye to the system after LAMP reaction, whether there is amplification product can be detected; The method of adding the nucleic acid dye is as follows: after the LAMP reaction system is prepared, a movable diaphragm that does not contact the reaction system is set at the upper end of the reaction tube, and the nucleic acid dye is dripped on the diaphragm, and then the lid is closed to react, and after the reaction is completed, the nucleic acid dye on the diaphragm is mixed into the system after the reaction by centrifugation, and the presence of an amplification product is determined by observing the color development result; The nucleic acid dye is calcein or SYBR Green I; The LAMP-based primer set for identifying codling moth consists of an F3 primer with a nucleotide sequence of SEQ ID NO: 2, a B3 primer with a nucleotide sequence of SEQ ID NO: 3, a FIP primer with a nucleotide sequence of SEQ ID NO: 4, and a BIP primer with a nucleotide sequence of SEQ ID NO:

5.

3. A portable kit for identifying codling moth based on visual LAMP technology, characterized in that: Comprising a LAMP primer set, wherein the LAMP primer set consists of an F3 primer having a nucleotide sequence of SEQ ID NO: 2, a B3 primer having a nucleotide sequence of SEQ ID NO: 3, a FIP primer having a nucleotide sequence of SEQ ID NO: 4, and a BIP primer having a nucleotide sequence of SEQ ID NO: 5; It also includes nucleic acid dye, Bst DNA large fragment polymerase, 10×ThermoPol buffer, 100mmol / L MgSO4, 10mmol / L dNTPs mixed solution, 5mol / L Betaine, movable diaphragm and constant temperature tool.

Images