Primer probe group, kit and method for simultaneously detecting multiple noctuids based on ERA

Abstract

The invention discloses a primer probe group, a kit and a method for simultaneously detecting multiple noctuids based on ERA (Endogenous Region Amplification). Each primer and probe can specifically amplify DNA (Deoxyribose Nucleic Acid) of spodoptera frugiperda, athetis lepigone, prodenia litura, beet armyworm, cotton bollworm, armyworm, white streak armyworm, argyrogramma agnata, noctuid litura, Trachea atriplicis and lepticia litura; on the basis, the detection kit and the detection method are formed. The detection method is high in detection sensitivity and specificity, and can be used for on-site rapid detection of various states of different noctuid pests.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapid molecular biological detection, and in particular relates to a primer, a kit and a method for detecting noctuid moths using a multi-tube combined method based on an enzymatic isothermal nucleic acid amplification technique (ERA). Background Art

[0002] Noctuidae pests are a major group of agricultural pests. Many important pests, including the armyworm (Mythimnaseparata), beet armyworm (Spodoptera exigua), fall armyworm (Spodoptera litura), cotton bollworm (Helicoverpa armigera), and fall armyworm (Spodoptera frugiperda), belong to this family. They are widely distributed and have a wide host range, preying on a variety of important crops, including corn, cotton, soybeans, wheat, sugar beets, and vegetables, severely impacting their yield and quality. First- and second-instar larvae infest crops day and night, feeding on entire leaves or creating holes and notches in them. After the third instar, larvae increase their appetite, feeding extensively on above-ground stems and leaves, leaving only stalks, often resulting in damage or even death to the entire crop plant. Currently, various methods exist for pest identification. Traditional identification relies on morphological differentiation, primarily relying on complete insect morphological features. However, this method has a low recognition rate for eggs, larvae, or incomplete specimens, requires specialized personnel, and is time-consuming. If timely and accurate identification is not possible, the appropriate prevention and control period is often missed, leading to pest outbreaks. For Noctuidae pests, not only is it difficult to identify eggs, larvae, or incomplete specimens, but their adult forms are also very similar, making identification equally difficult.

[0003] Among the currently developed molecular detection methods for identifying noctuid pests, PCR technology requires complex instrumentation and strict temperature cycling control, making it impractical to implement in the field or at ports. LAMP technology relies on the design of six primers, making the design of primers for multiple species detection exponentially more difficult. Furthermore, nucleic acid dyes can inhibit amplification, affecting detection stability. Enzymatic recombinant isothermal amplification (ERA) is widely used in pathogen detection because it requires no denaturation and can be performed at room temperature. However, no ERA-based technology for detecting noctuid pests has been developed. Summary of the Invention

[0004] The object of the present invention is to provide a primer probe set for simultaneously detecting multiple noctuid moths based on ERA, which can amplify DNA samples of multiple noctuid moths without mutual interference.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A primer-probe set for simultaneous detection of multiple noctuid moths based on ERA, comprising any two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following primers and probes for detecting noctuid moths:

[0007] Primers and probe 1 for detecting Spodoptera frugiperda: Primer F: 5'-GGTGGTAGTTCAGTAGATTTAGCTATTTTC-3', SEQ ID No. 1; Primer R: 5'-ATCAGTAAGTAATATAGTAATAGCTCCAGC-3', SEQ ID No. 2; Probe PBR: TTATTTATTTGAGCTGTAGGTATTACCGCA (FAM-dT) (THF) (BHQ1-dT) TTAT TATTATTAT (C3-SPACER), SEQ ID No. 3;

[0008] Primers and probe 2 for detecting Spodoptera exigua: Primer F: 5'-TTGCTCATGGTGGAAGATCAGTAGATTTAG-3', SEQ ID No. 4; Primer R: 5'-GAATAGGGTCACCTCCTCCTGCTGGATCAA-3', SEQ ID No. 5; Probe PBR: CTCCAGCTAAAACAGGTAAAGATAATAA(FAM-dT)A(THF)(BHQ1-dT)AAA AATGCAGTAAT(C3-SPACER), SEQ ID No. 6;

[0009] Primers and probe 3 for detecting Spodoptera litura: Primer F: 5'-TTGCTCATGGTGGAAGATCAGTAGATTTAG-3', SEQ ID No. 7; Primer R: 5'-GAATAGGGTCACCTCCTCCTGCTGGATCAA-3', SEQ ID No. 8; Probe PBR: CTCCAGCTAAAACAGGTAAAGATAATAA(FAM-dT)A(THF)(BHQ1-dT)AAA AATGCAGTAAT(C3-SPACER), SEQ ID No. 9;

[0010] Primers and probe 4 for detecting beet armyworm: Primer F: 5'-ATTTTTTCTTTACATTTAGCTGGAATTTCT-3', SEQ ID No. 10; Primer R: 5'-TTCGGTCTGTAAGTAATATAGTAATAGCTC-3', SEQ ID No. 11; Probe PBR: TAGTGATAGTAATAATAAAAAAGCAGTAAT(FAM-dT)C(THF)(BHQ1-dT)AC AGCTCAAACA(C3-SPACER), SEQ ID No. 12;

[0011] Primers and probe 5 for detecting cotton bollworm: Primer F: 5'-TTCCCCCTTCTTTAACTTTACTTATTTCAA-3', SEQ ID No. 13; Primer R: 5'-CACCTGCTAAAACTGGTAATGATAATAATA-3', SEQ ID No. 14; Probe PBR: ATTGCTCCTAAAATAGATGAGATTCCAGC(FAM-dT)A(THF)A(BHQ1-dT)GT AAAGAAAAAA(C3-SPACER), SEQ ID No. 15;

[0012] Primers and probe 6 for detecting Mythimna separata: Primer F: 5'-CTTTCATCAAATATTGCCCATGGAGGTAGA-3', SEQ ID No. 16; Primer R: 5'-TAAACTTCTGGATGACCAAAAAATCAAAAT-3', SEQ ID No. 17; Probe PBR: TACCTTTATTTATTTGAGCTGTTGGGAT(FAM-dT)A(THF)(BHQ1-dT)GCATT TTTACTATT(C3-SPACER), SEQ ID No. 18;

[0013] Primers and probe 7 for detecting Spodoptera littoralis: Primer F: 5'-TTTTGACTTCTTCCCCCATCTTTAACATTA-3', SEQ ID No. 19; Primer R: 5'-TACAGGTAAAGATAATAATAAAAGGAAAGC-3', SEQ ID No. 20; Probe PBR: GGTAAACCGTTCATCCAGTTCCTGCTCCAT(FAM-dT)(THF)(BHQ1-dT)CTACAATTCTTCT(C3-SPACER), SEQ ID No. 21;

[0014] Primers and probe 8 for detecting Spodoptera: Primer F: 5'-TCTTTTATTATTATCCTTACCAGTATTAGC-3', SEQ ID No. 22; Primer R: 5'-TAAAATGTAAACTTCAGGATGACCAAAAAA-3', SEQ ID No. 23; Probe PBR: CTCCTCCTCCGGCAGGGTCAAAAAAAGAAG(FAM-dT)(THF)(BHQ1-dT)TTAAATTACGATC(C3-SPACER); SEQ ID No. 24;

[0015] Primers and probe 9 for detecting Spodoptera exigua: Primer F: 5'-TTGATTACTTCCACCTTCTATTACTTTATT-3', SEQ ID No. 25; Primer R: 5'-AAATAGCTAAATCTACTGATCTTCCTCCAT-3', SEQ ID No. 26; Probe PBR: ATAAACTGTTCATCCTGTTCCTGCTCCAT (FAM-dT) (THF) (BHQ1-dT) CTACA ATTCTTCTT (C3-SPACER), SEQ ID No. 27;

[0016] Primers and probe 10 for detecting Spodoptera: Primer F: 5'-TTTGATTACTTCCCCCCTCTTTAACTCTGT-3', SEQ ID No. 28; Primer R: 5'-AGCTCCAGCTAATACAGGTAGAGATAATAA-3', SEQ ID No. 29; Probe PBR: CATTTTTCATTACACTTAGCAGGTATT(FAM-dT)C(THF)(BHQ1-dT)CTATT TTAGGAGCA(C3-SPACER), SEQ ID No. 30;

[0017] Primers and probe 11 for detecting Spodoptera exigua: Primer F: 5'-TTTTTGACTTCTCCCCCCATCATTAACTTT-3', SEQ ID No.31; Primer R: 5'-GGTAAAGAAAGTAAAAGAAGAAATGCTGTA-3', SEQ ID No.32; Probe PBR: ATCTACAGAGCTACCTCCATGAGCGATAT(FAM-dT)A(THF)A(BHQ1-dT)GAAAGTGGGGGG(C3-SPACER), SEQ ID No.33.

[0018] Another object of the present invention is to provide a kit for simultaneously detecting multiple noctuid moths based on ERA, comprising: an ERA amplification reagent; the primer probe set according to claim 1; an activator; and ultrapure water.

[0019] Optionally, the activator is a magnesium acetate solution, and the primers and probes in the primer-probe set are lyophilized together with the amplification reagent into microspheres and pre-packed into independent cryopreservation tubes according to different noctuid species.

[0020] Optionally, the kit uses a 50 μL reaction system, 47 μL ultrapure water, 2 μL activator, and 1 μL nucleic acid sample to be tested; the fluorescent ERA amplification time of the kit is within 20 minutes.

[0021] A third object of the present invention is to provide a method for detecting noctuid moths, comprising the steps of:

[0022] Step S1: taking several cryovials containing primers and probes, adding ultrapure water to dissolve the freeze-dried primers and probes and amplification reagents, adding the test samples to each cryovial, shaking and mixing, and centrifuging; the primers and probes are the primers and probes of claim 1, the probes and primers in each cryovial are targeted to a certain species of Spodoptera in claim 1, and the test samples are genomic DNA extracted from eggs, larvae, pupae or adults of one or more species of Spodoptera;

[0023] Step S2: Add an activator to the cryopreservation tube, shake and mix, and centrifuge to obtain a sample group. Ultrapure water is used as a blank control group.

[0024] Step S3: placing the reaction tube in a fluorescence thermostat amplification instrument or a fluorescence quantitative PCR instrument for amplification. Step S3: placing the reaction tube in a fluorescence thermostat amplification instrument or a fluorescence quantitative PCR instrument for amplification. The amplification program is 35-42°C, the reaction does not exceed 20 minutes, and the FAM channel fluorescence value is collected every 30 seconds.

[0025] Step S4: analyzing whether the sample to be tested contains the Spodoptera moth corresponding to each of the cryopreservation tubes according to the positive or negative test results.

[0026] Optionally, in step S4, the judgment criteria are:

[0027] A positive result was determined if the fluorescence signal value exceeded 3 times the baseline standard deviation within 20 minutes and the Ct value was less than 15 minutes. A negative result was determined if the fluorescence signal value was low and there was no Ct value or the Ct value was greater than 15 minutes. If a positive result appeared in a cryotube, it indicated that the sample to be tested contained the corresponding Spodoptera moth of the cryotube. If a negative result appeared in a cryotube, it indicated that the sample to be tested did not contain the corresponding Spodoptera moth of the cryotube.

[0028] Preferably, the amplification process is performed at 40°C and the reaction time is 20 minutes.

[0029] The present invention has the following beneficial effects:

[0030] (1) Based on the characteristics and differential sequence regions of the COI genes of various Noctuidae insects, multiple ERA detection primers and probes were designed and screened. When amplifying mixed samples containing DNA of multiple Noctuidae insects, the selected primers all have their own specificity, that is, they only amplify the DNA of a certain Noctuidae insect, but not the DNA of other Noctuidae insects, which creates creative conditions for the simultaneous detection of multiple Noctuidae insects.

[0031] (2) The rapid combined detection kit for multiple noctuid moths can complete nucleic acid amplification at a constant temperature of 35°C-42°C without the need for denaturation of template DNA. The detection can be completed within 20 minutes. It is easy to operate and does not require professional personnel and complex instruments and equipment. The detection sensitivity and specificity are high. It can not only be used for on-site rapid detection of various insect stages of different noctuid pests, but also provides a usable rapid identification method for on-site identification of pests. It is particularly suitable for use at the grassroots level and can bring great convenience to customs, ports and other quarantine departments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a specific detection diagram of the primers and probes for the fall armyworm of the present invention, 1: fall armyworm; 2-12: armyworm, two-spotted moth, cotton bollworm, black cutworm, Spodoptera litura, white-striped armyworm, Spodoptera exigua, Spodoptera spp., thin silver ingot moth, and silver stripe moth, in order.

[0033] Figure 2 This is a specific detection diagram of the primers and probes of the armyworm of the present invention, 1: armyworm; 2-12: fall armyworm, two-spotted armyworm, cotton bollworm, black cutworm, Spodoptera litura, white-striped armyworm, Spodoptera exigua, Spodoptera spp., thin silver ingot armyworm, and silver stripe armyworm, respectively.

[0034] Figure 3 This is a specific detection diagram of the primers and probes of the two-spotted armyworm of the present invention, 1: two-spotted armyworm; 2-12: armyworm, fall armyworm, cotton bollworm, black cutworm, Spodoptera litura, white-striped armyworm, Spodoptera exigua, Spodoptera pusillus, Spodoptera slendersii, and Spodoptera silver moth.

[0035] Figure 4 This is a specific detection diagram of the primers and probes for cotton bollworm of the present invention, 1: cotton bollworm; 2-12: armyworm, two-spotted armyworm, fall armyworm, black cutworm, Spodoptera litura, white-striped armyworm, Spodoptera exigua, beet armyworm, Spodoptera faecalis, Spodoptera tenuifolia, and Spodoptera argentipes, in order.

[0036] Figure 5This is a specific detection diagram of the primers and probes for Spodoptera litura of the present invention, 1: Spodoptera litura; 2-12: armyworm, two-spotted moth, cotton bollworm, black cutworm, fall armyworm, white-striped moth, Spodoptera exigua, beet armyworm, Spodoptera fumigatus, thin silver ingot moth, and silver stripe moth, in order.

[0037] Figure 6 This is a specific detection diagram of the primers and probes of the white-striped armyworm of the present invention, 1: white-striped armyworm; 2-12: armyworm, two-spotted armyworm, cotton bollworm, black cutworm, Spodoptera litura, fall armyworm, Spodoptera exigua, Spodoptera pusillus, Spodoptera slendersii, and Spodoptera argentipes.

[0038] Figure 7 This is a specific detection diagram of the primers and probes for the Spodoptera exigua of the present invention, 1: Spodoptera exigua; 2-12: respectively, armyworm, two-spotted moth, cotton bollworm, black cutworm, Spodoptera litura, white-striped moth, fall armyworm, beet armyworm, Spodoptera exigua, Spodoptera exigua, Spodoptera slendersalis, and Spodoptera silver moth.

[0039] Figure 8 This is a specific detection diagram of the primers and probes for beet armyworm of the present invention, 1: beet armyworm; 2-12: armyworm, two-spotted moth, cotton bollworm, black cutworm, Spodoptera litura, white-striped moth, Spodoptera frugiperda, Spodoptera slendersii, Spodoptera slendersii, and Spodoptera slendersii.

[0040] Figure 9 This is a specific detection diagram of the primers and probes of the Spodoptera exigua of the present invention, 1: Spodoptera exigua; 2-12: respectively, armyworm, two-spotted moth, cotton bollworm, black cutworm, Spodoptera litura, white-striped moth, Spodoptera exigua, fall armyworm, Spodoptera exigua, and silver-striped moth.

[0041] Figure 10 This is a specific detection diagram of the primers and probes of the thin silver ingot armyworm of the present invention, 1: thin silver ingot armyworm; 2-12: armyworm, two-spotted moth, cotton bollworm, black cutworm, Spodoptera litura, white-striped armyworm, Spodoptera exigua, Spodoptera frugiperda, Spodoptera frugiperda, and Spodoptera frugiperda.

[0042] Figure 11 This is a specific detection diagram of the primers and probes for the silver-striped armyworm of the present invention, 1: silver-striped armyworm; 2-12: armyworm, two-spotted armyworm, cotton bollworm, black cutworm, armyworm, white-striped armyworm, black cutworm, beet armyworm, black cutworm, thin silver ingot armyworm, and fall armyworm.

[0043] Figure 12This is the anti-interference detection diagram of the primers and probes of the fall armyworm of the present invention, 1: single fall armyworm DNA template; 2: mixed template of 12 fall armyworm DNAs; 3: mixed template of the DNAs of the other fall armyworms except fall armyworm; 4: CK (water is used instead of DNA template, the same below).

[0044] Figure 13 This is an anti-interference detection diagram of the primers and probes of the armyworm of the present invention, 1: single armyworm DNA template; 2: mixed template of 12 species of noctuid DNA; 3: mixed template of DNA of other noctuids except the armyworm; 4: CK.

[0045] Figure 14 This is an anti-interference detection diagram of the primers and probes for Spodoptera exigua of the present invention, 1: single Spodoptera exigua DNA template; 2: mixed template of 12 species of Spodoptera DNA; 3: mixed template of DNA of other Spodoptera except Spodoptera exigua; 4: CK.

[0046] Figure 15 This is an anti-interference detection diagram of the primers and probes for cotton bollworm of the present invention, 1: single cotton bollworm DNA template; 2: mixed template of 12 species of noctuid DNA; 3: mixed template of DNA of other noctuids except cotton bollworm; 4: CK.

[0047] Figure 16 This is an anti-interference detection diagram of the primers and probes for Spodoptera litura of the present invention, 1: single Spodoptera litura DNA template; 2: mixed template of 12 species of Spodoptera DNA; 3: mixed template of DNA of other Spodoptera except Spodoptera litura; 4: CK.

[0048] Figure 17 This is an anti-interference detection diagram of the primers and probes of the white-striped moth of the present invention, 1: single white-striped moth DNA template; 2: mixed template of 12 species of moth DNA; 3: mixed template of DNA of moths other than white-striped moth; 4: CK.

[0049] Figure 18 This is a diagram of the anti-interference detection of the primers and probes for Spodoptera exigua of the present invention, 1: single Spodoptera exigua DNA template; 2: mixed template of 12 species of Spodoptera DNA; 3: mixed template of DNA of other Spodoptera except Spodoptera exigua; 4: CK.

[0050] Figure 19 This is an anti-interference detection diagram of the primers and probes for beet armyworm of the present invention, 1: single beet armyworm DNA template; 2: mixed template of 12 species of armyworm DNA; 3: mixed template of DNA of other armyworms except beet armyworm; 4: CK.

[0051] Figure 20This is an anti-interference detection diagram of the primers and probes of the Spodoptera argentatum of the present invention, 1: single Spodoptera argentatum DNA template; 2: mixed template of 12 species of Spodoptera DNA; 3: mixed template of DNA of other Spodoptera except Spodoptera argentatum; 4: CK.

[0052] Figure 21 This is an anti-interference detection diagram of the primers and probes of the Spodoptera tenuifolia of the present invention, 1: a single Spodoptera tenuifolia DNA template; 2: a mixed template of 12 species of Spodoptera DNA; 3: a mixed template of DNAs of other Spodoptera except Spodoptera tenuifolia; 4: CK.

[0053] Figure 22 This is an anti-interference detection diagram of the primers and probes of Spodoptera argentea of ​​the present invention, 1: single Spodoptera argentea DNA template; 2: mixed template of 12 species of Spodoptera DNA; 3: mixed template of DNA of other Spodoptera except Spodoptera argentea; 4: CK. DETAILED DESCRIPTION

[0054] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. The ERA amplification reagent in the examples is a product of Suzhou Xianda Gene Technology Co., Ltd.

[0055] The present invention uses the differential partial sequence regions of the COI genes of the fall armyworm, the two-spotted moth, the cutworm, the beet armyworm, the cotton bollworm, the armyworm, the white-striped armyworm, the silver-striped armyworm, the black-striped armyworm, the white-striped armyworm, the black-striped armyworm, the white-striped armyworm, and the thin-silvered armyworm as detection targets, designs ERA detection primers and probes, and pre-loads them into 11 independent cryopreservation tubes through freeze-drying technology, thereby establishing a method for the joint and synchronous detection of multiple noctuid pests based on isothermal amplification, which is illustrated below through specific examples.

[0056] Example 1 Design and synthesis of primers and probes for ERA detection of various Noctuidae pests

[0057] Based on the differential sequence regions of the COI genes of Spodoptera frugiperda, Spodoptera litura, Spodoptera litura, Spodoptera exigua, Helicoverpa armigera, Armyworm, Spodoptera striata, Spodoptera argentipes, Spodoptera falciparum, Spodoptera striata, Spodoptera striata, and Spodoptera slendersinus, three pairs of primers and one probe were designed for each species for screening. The designed primers and probes are shown in Table 1:

[0058] Table 1 Primer and probe sequence information

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] In these probes, FAM-dT serves as the FAM fluorescent reporter, and BHQ1-dT serves as the BHQ1 fluorescence quencher. THF (tetrahydrofuran) serves as the cleavage site for the exonuclease exo. C3-space: C3 occlusion blocks extension, hindering the exonuclease and polymerase. These primers and probes were synthesized by General Biotech (Anhui) Co., Ltd.

[0065] Example 2 ERA Detection Method for Noctuidae Pests

[0066] Using the genomic DNA of the Spodoptera moth sample as a template, perform an ERA reaction using the primers and probe designed in step 1 to obtain the ERA amplification product and detect the fluorescence signal intensity. The specific steps are as follows:

[0067] The ERA detection system is 50 μL, including 2.1 μL of upstream and downstream primers (10 μM), 0.6 μL of probe (10 μM), and 1 μL of DNA template. The rest is made up to 48 μL with ddH2O. The premix of each sample is prepared according to the following ERA reaction system composition, thoroughly shaken and briefly centrifuged.

[0068] Components Sample volume per tube / μL Forward primer (10 μM) 2.1 Reverse primer (10 μM) 2.1 Fluorescent probe (10 μM) 0.6 DNA template 1 <![CDATA[ddH2O]]> 22.2 volume 48

[0069] For each sample, transfer 48 μL of the premix to each tube of lyophilized microspheres using ERA fluorescent nucleic acid amplification reagent, manufactured by Suzhou Xinda Gene Technology Co., Ltd. Vortex to mix until the microspheres are resuspended and briefly centrifuge. Then, add 2 μL of the activator, magnesium acetate solution, to the cap of the reaction tube. Carefully secure the cap and briefly centrifuge to incorporate the activator into the premix. Briefly mix and quickly centrifuge. Immediately place the tube in a fluorescence quantitative PCR instrument or fluorescence amplification detector and initiate the reaction at 40°C for 20 minutes. Fluorescence values ​​in the FAM channel are collected every 30 seconds.

[0070] If the ERA amplification product meets the following conditions: an obvious amplification curve appears within 20 minutes of ERA amplification (the fluorescence signal value exceeds 3 times the standard deviation of the baseline and the Ct value is less than 15 minutes), it is positive; if there is no obvious amplification curve (the fluorescence signal value is low and there is no Ct value or the Ct value is greater than 15 minutes), it is negative.

[0071] Example 3 Screening experiment of primers and probes for ERA detection of various noctuid pests

[0072] The sequence information of each primer and probe is shown in the table in Example 1. Before the experiment, the genomic DNA of each moth was diluted to 10 -2 ng / μL as the template to be tested.

[0073] Primer screening: Each species has three upstream primers F1 / F2 / F3, three downstream primers R1 / R2 / R3, and a probe. During screening, F1 is paired with R1 / R2 / R3 respectively, and the probe is added to prepare a premix. A negative control (i.e., water is added as a template) and a positive control (i.e., the corresponding Spodoptera genotype DNA is used as a template) are used. Using the above reaction system, a pair of primers with almost no change in the fluorescence value of the negative control and a clear fluorescence value curve of the positive control is selected, such as F1 / R3. R3 is paired with the remaining two upstream primers, i.e., F2 / F3, and the experiment is repeated. In this way, primer pairs with high amplification efficiency and small Ct value are screened for each species, as shown in Table 2.

[0074] Table 2 Screened primers and probes

[0075]

[0076]

[0077] Example 4 Specific detection experiment of primers and probes for single moth samples

[0078] The sequence information of each primer and probe is shown in Table 2 of Example 3. Before the experiment, the genomic DNA of each moth was diluted to 10 -2 ng / μL as the template to be tested.

[0079] Specificity detection experiment of a single noctuid sample: Each set of primers and probes in Table 2 was used to amplify the 11 noctuid samples described in Table 2, and two were tested in parallel, where the noctuid genomic DNA template corresponding to the primers and probes used was used as the positive control, and the rest were negative controls. If the positive control shows an obvious amplification curve (the fluorescence signal value exceeds 3 times the standard deviation of the baseline and the Ct value is <15min), and the negative control has no obvious amplification curve (the fluorescence signal value is low and there is no Ct value or the Ct value is >15min), the specificity is qualified. After testing, the primers and probes in Table 2 only produce obvious amplification curves for the corresponding species, and have no obvious amplification for other closely related species, and have good specificity. The specific results are as follows Figures 1 to 11 shown.

[0080] Example 5 Specific detection experiment of primers and probes for mixed samples of 11 species of noctuid moths

[0081] Each set of primers and probes in Table 2 was used as a positive control for the mixed template of genomic DNA of the 11 species of noctuids described in Table 2, and the primers and probes were used as positive controls with the corresponding single noctuid genomic DNA; each primer and probe in Table 2 was used as a negative control for the mixed template of genomic DNA of the 10 species of noctuids other than the noctuids corresponding to the primers and probes, and water was used to replace the noctuid genomic DNA as a template as a negative control, and the two were tested in parallel. If the positive control shows an obvious amplification curve (the fluorescence signal value exceeds 3 times the standard deviation of the baseline and the Ct value is less than 15min), and the negative control has no obvious amplification curve (the fluorescence signal value is low and there is no Ct value or the Ct value is greater than 15min), it means that the primers and probes have good specificity for the 11 noctuid samples. After testing, the primers and probes described in Table 2 have good specificity for the mixed sample of 11 species of noctuids. The specific experimental results are as follows. Figures 12 to 22 The present invention also designed three primers and probes for the cutworm Agrotis spp. However, according to the methods of Examples 4 and 5, it was found that the primers and probes designed for the cutworm Agrotis spp. also amplified other noctuid moths and could not meet the specificity standard. Therefore, these primers and probes were not included in the study.

Claims

1. A primer probe set for simultaneous detection of multiple noctuid moths based on ERA, characterized in that Any two, three, four, five, six, seven, eight, nine, ten, or all eleven of the following primers and probes for detecting Spodoptera moth are selected: Primers and probe 1 for detecting Spodoptera frugiperda: the upstream primer sequence is shown in SEQ ID No. 1, the downstream primer sequence is shown in SEQ ID No. 2, and the probe sequence is shown in SEQ ID No. 3; Primers and probe 2 for detecting Spodoptera exigua: the upstream primer sequence is shown in SEQ ID No. 4, the downstream primer sequence is shown in SEQ ID No. 5, and the probe sequence is shown in SEQ ID No. 6; Primers and probe 3 for detecting Spodoptera litura: the upstream primer sequence is shown in SEQ ID No. 7, the downstream primer sequence is shown in SEQ ID No. 8, and the probe sequence is shown in SEQ ID No. 9; Primers and probe 4 for detecting Spodoptera exigua: the upstream primer sequence is shown in SEQ ID No. 10, the downstream primer sequence is shown in SEQ ID No. 11, and the probe sequence is shown in SEQ ID No. 12; Primers and probe 5 for detecting cotton bollworm: the upstream primer sequence is shown in SEQ ID No. 13, the downstream primer sequence is shown in SEQ ID No. 14, and the probe sequence is shown in SEQ ID No. 15; Primers and probe 6 for detecting Mythimna separata: the upstream primer sequence is shown in SEQ ID No. 16, the downstream primer sequence is shown in SEQ ID No. 17, and the probe sequence is shown in SEQ ID No. 18; Primers and probe 7 for detecting Spodoptera littoralis: the upstream primer sequence is shown in SEQ ID No. 19, the downstream primer sequence is shown in SEQ ID No. 20, and the probe sequence is shown in SEQ ID No. 21; Primers and probe 8 for detecting Spodoptera: the upstream primer sequence is shown in SEQ ID No. 22, the downstream primer sequence is shown in SEQ ID No. 23, and the probe sequence is shown in SEQ ID No. 24; Primers and probe 9 for detecting Spodoptera exigua: the upstream primer sequence is shown in SEQ ID No. 25, the downstream primer sequence is shown in SEQ ID No. 26, and the probe sequence is shown in SEQ ID No. 27; Primers and probe 10 for detecting Spodoptera: the upstream primer sequence is shown in SEQ ID No. 28, the downstream primer sequence is shown in SEQ ID No. 29, and the probe sequence is shown in SEQ ID No. 30; Primers and probe 11 for detecting Spodoptera tenella: the upstream primer sequence is shown in SEQ ID No.31, the downstream primer sequence is shown in SEQ ID No.32, and the probe sequence is shown in SEQ ID No.

33.

2. A kit for simultaneous detection of multiple noctuid moths based on ERA, characterized in that include: ERA amplification reagent; The primer probe set according to claim 1; Activator; Ultrapure water.

3. The kit according to claim 2, wherein: The activator is a magnesium acetate solution. The primers and probes in the primer-probe set are freeze-dried together with the ERA amplification reagent to form microspheres and are pre-packed into independent cryopreservation tubes according to different noctuid species.

4. The kit according to claim 3, wherein: The kit uses a 50 μL reaction system, 47 μL ultrapure water, 2 μL activator, and 1 μL nucleic acid sample to be tested; the fluorescent ERA amplification time of the kit is within 20 minutes.

5. A method for detecting noctuid moths, characterized in that The steps include: Step S1: taking several cryovials pre-filled with primers, probes, and amplification reagents, adding ultrapure water to dissolve the freeze-dried primers, probes, and amplification reagents, adding the test samples to each cryovial, shaking and mixing, and centrifuging; the primers and probes are the primers and probes of claim 1, the probes and primers in each cryovial are each targeted to a certain species of Spodoptera moth of claim 1, and the test samples are genomic DNA extracted from eggs, larvae, pupae, or adults of one or more species of Spodoptera moth; Step S2: Add an activator to the cryopreservation tube, shake and mix, and centrifuge to obtain a sample group. Ultrapure water is used as a blank control group. Step S3: Place the reaction tube in a fluorescence thermostat amplification instrument or a fluorescence quantitative PCR instrument for amplification at 35-42°C for no more than 20 minutes, and collect the FAM channel fluorescence value every 30 seconds; Step S4: analyzing whether the sample to be tested contains the Spodoptera moth corresponding to each of the cryopreservation tubes according to the positive or negative test results.

6. The method for detecting noctuid moths according to claim 5, characterized in that: In step S4, the judgment criteria are: A positive result was determined if the fluorescence signal value exceeded 3 times the baseline standard deviation within 20 minutes and the Ct value was less than 15 minutes. A negative result was determined if the fluorescence signal value was low and there was no Ct value or the Ct value was greater than 15 minutes. If a positive result appeared in a cryotube, it indicated that the sample to be tested contained the corresponding Spodoptera moth of the cryotube. If a negative result appeared in a cryotube, it indicated that the sample to be tested did not contain the corresponding Spodoptera moth of the cryotube.

7. The detection method according to claim 5, wherein The amplification procedure is 40° C. and the reaction time is 20 min.

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