Multiplex PCR (Polymerase Chain Reaction) primer and method for rapidly identifying thrips insect varieties

Abstract

The invention belongs to the technical field of molecular identification of insects, and particularly relates to multiple PCR (Polymerase Chain Reaction) primers and a method for rapidly identifying thrips insect varieties. The invention discloses a multiple PCR primer and a method for rapidly identifying the variety of thrips, the identification primer and a multiple PCR system can rapidly, effectively and accurately identify the three types of thrips, and the sizes of PCR product strips of tobacco thrips, comb flower thrips and common large thrips are 104 bp, 237 bp and 439 bp respectively. The detection limit of the sensitive tobacco thrips is 500 copies, and the detection limits of the sensitive tobacco thrips and the sensitive common large thrips are both 100 copies. Compared with common PCR which can only identify one type of thrips at a time, the multiplex PCR system can complete identification of various types of thrips at a time, has the advantages of being easy to operate, low in cost, capable of saving time and the like, has very high value in the aspect of field thrips detection, and can be widely applied to the fields of plant protection and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of insect molecular identification, and particularly relates to a multiplex PCR primer and method for rapidly identifying Thripidae insect species. Background Art

[0002] Thrips are a type of pest with tiny body size and strong hiding ability. They belong to the class Insecta, order Thysanoptera, family Thripidae. They are widely distributed in tropical and subtropical regions around the world and are relatively common in various parts of my country. Thrips mainly live on crops of the Cucurbitaceae, Leguminosae, Cruciferae, and Solanaceae families. Because their adult insects are afraid of strong light, they often concentrate on causing harm in backlit places. They will only move on the surface of host plants when the light intensity is weak, such as on cloudy days, in the early morning, and in the evening. Thrips are small in size, usually between 1 and 2 mm in length, with bristles on the rear edge of the prothorax, slender and transparent wings with many slender hairs around the edges, and rasping mouthparts that suck the juice by rasping the plant epidermal tissue, harming tender shoots and leaves, causing them to curl and harden, and the heart leaves cannot unfold normally, forming deformities; after harming young fruits, scars will form on the feeding parts as the fruits swell, and the scars will continue to expand with the expansion of the fruits, showing different shapes and degrees of cork, and in severe cases, even causing fruit drop, seriously affecting quality. In addition to directly harming crops, thrips can also indirectly spread viral diseases. Due to the characteristics of many species, small size and easy to develop drug resistance, thrips have rapidly developed into a bottleneck problem restricting vegetable production in Hainan.

[0003] Traditional identification of Thripidae insect species mainly relies on morphological identification, which is mainly judged by observing their body shape, color, antennae, wing shape, mouthpart structure and leg characteristics. However, in cases where morphological identification experience is relatively weak and environmental conditions are harsh, morphological identification cannot distinguish them well and there is a risk of misjudgment, resulting in misjudgment of thrips species, which in turn affects the effect and progress of field vegetable pest control. Therefore, a method based on molecular biology technology that can quickly and accurately identify three Thripidae insects and their mixtures is needed. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide a multiplex PCR detection primer for detecting thrips.

[0005] The purpose of the second aspect of the present invention is to provide the application of the multiplex PCR detection primers of the first aspect of the present invention.

[0006] The third aspect of the present invention aims to provide a kit for detecting thrips.

[0007] The fourth aspect of the present invention aims to provide a method for identifying thrips.

[0008] The fifth aspect of the present invention aims to provide a universal primer for detecting thrips.

[0009] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a multiplex PCR detection primer for detecting thrips.

[0011] In some embodiments of the present invention, the thrips include Thrips tabaci, Frankliniella schultzei and Megalurothrips usitatus.

[0012] In some embodiments of the present invention, the multiplex PCR detection primers include specific primers for detecting Thrips tabaci, Thrips oleraceus and Thrips thrips;

[0013] In some embodiments of the present invention, the specific primer sequences for detecting Thrips tabaci are shown in SEQ ID NOs: 3 and 4.

[0014] In some embodiments of the present invention, the specific primer sequences for detecting thrips spp. are shown in SEQ ID NOs: 5 and 6.

[0015] In some embodiments of the present invention, the specific primer sequences for detecting common Thrips are shown in SEQ ID NOs: 7 and 8.

[0016] The second aspect of the present invention provides the use of the multiplex PCR detection primers described in the first aspect of the present invention in at least one of (a1) to (a6):

[0017] (a1) Identification of Thrips tabaci;

[0018] (a2) preparing products for identifying Thrips tabaci;

[0019] (a3) Identification of thrips;

[0020] (a4) preparing a product for identifying Thrips spp.;

[0021] (a5) Identification of common Macrothrips;

[0022] (a6) Preparation of a product for identifying common Thrips.

[0023] In some embodiments of the present invention, the product includes at least one of a reagent, a kit, a test paper, a chip, and a system.

[0024] The third aspect of the present invention provides a kit for detecting thrips, the kit comprising the multiplex PCR detection primers described in the first aspect of the present invention.

[0025] In some embodiments of the present invention, the kit further comprises Taq DNA polymerase, dNTP, PCR buffer, Mg 2+ At least one of .

[0026] In some embodiments of the present invention, the kit further comprises a negative control standard and a positive control standard.

[0027] A fourth aspect of the present invention provides a method for identifying thrips, characterized in that it comprises the following steps:

[0028] 1) using the multiplex PCR detection primers described in the first aspect of the present invention or the kit described in the third aspect of the present invention to perform PCR amplification on the sample to be tested;

[0029] 2) Analyze the size of the amplified product and identify the sample type based on the size of the amplified product band.

[0030] In some embodiments of the present invention, the sample to be tested includes genomic DNA of an insect sample.

[0031] In some embodiments of the present invention, the reaction conditions of the PCR amplification are: based on a 20 μL reaction system, including 10-100 ng of DNA template, multiple PCR primers with a concentration of 10 μM, 0.4 μL of each, 10 μL of 2×PCR Buffer, and the rest is ddH2O.

[0032] In some embodiments of the present invention, the PCR reaction conditions are: pre-deformation at 95°C for 8 to 12 min; deformation at 95°C for 20 to 40 s; annealing at 55 to 57°C for 50 to 70 s, extension at 71 to 73°C for 20 to 40 s, for a total of 30 to 40 cycles; extension at 71 to 73°C for 6 to 10 min.

[0033] Specifically, the PCR reaction conditions are: pre-deformation at 95°C for 10 min; deformation at 95°C for 30 s; annealing at 56°C for 1 min, extension at 72°C for 30 s, for a total of 35 cycles; and extension at 72°C for 8 min.

[0034] In some embodiments of the present invention, the detection method is agarose gel electrophoresis (2%).

[0035] In some embodiments of the present invention, the thrips include Thrips tabaci, Thrips spp. and Thrips spp.

[0036] In some embodiments of the present invention, when a 104 bp band is present in the amplified product, it is determined that the sample contains Thrips tabaci;

[0037] When the amplified product has a 237 bp band, it is judged that the sample contains Thrips punctatus;

[0038] When a 439 bp band is present in the amplified product, it is determined that the sample contains Macrothrips cyprinidae.

[0039] A fifth aspect of the present invention provides a universal primer for detecting thrips, wherein the sequence of the primer is shown in SEQ ID NO: 1, 2.

[0040] The beneficial effects of the present invention are:

[0041] The present invention uses the COI gene sequence from the mitochondrial genome of Thripidae insects to design and synthesize three pairs of specific PCR primers for different Thripidae insect species, and uses the primers to establish an accurate, fast and economical multiple PCR reaction, realizing a new method for quickly identifying three Thripidae insect species using molecular biological methods, which has high value in field thrips detection. The invention can be widely used in fields such as plant protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0043] Figure 1 The primer-specific PCR electrophoresis detection diagram is as follows: M-100bp marker, lanes 1-6: primers THtab-F / THtab-R amplify 1-Tanzania tabaci, 2-Comb flower thrips, 3-Common giant thrips, 4-Tube thrips, 5-Apple thrips, 6-Palm thrips; lanes 7-12:

[0044] Primers FRsch-F / FRsch-R amplified 7-comb flower thrips, 8-tobacco thrips, 9-common big thrips, 10-tube thrips, 11-apple thrips,

[0045] 12- palmitothrips; lanes 13-18: MEusi-F / MEusi-R amplified 13- common giant thrips, 14- tobacco thrips, 15- comb flower thrips, 16- tube thrips, 17- apple thrips, 18- palmitothrips.

[0046] Figure 2 Schematic diagram of the multiplex PCR electrophoresis results of single and multiple individuals of three Thripidae insects: Thripidae insect species from left to right are: M-100bp marker, 1-Tanzania tabaci, Thrips fasciatus, and common Thrips mixed sample, 2-Tanzania tabaci, 3-Tanzania fasciatus, 4-common Thrips, 5-Tropicrin, 6-Apple Thrips, 7-Palm Thrips, and "-" blank control.

[0047] Figure 3 Schematic diagram of the sensitivity electrophoresis results of multiple PCR for Thrips tabaci, M-100bp marker, 1-single-end sample DNA without dilution, 10-DNA sample 10-fold dilution, 100-DNA sample 100-fold dilution, 500-DNA sample 500-fold dilution, 1000-DNA sample 1000-fold dilution, 2000-DNA sample 2000-fold dilution.

[0048] Figure 4 The figure is a schematic diagram of the sensitivity electrophoresis results of multiple PCR for Thrips punctatus, M-100bp marker, 1-single-end sample DNA without dilution, 10-DNA sample 10-fold dilution, 100-DNA sample 100-fold dilution, 500-DNA sample 500-fold dilution,

[0049] The 1000-DNA sample was diluted 1000-fold, and the 2000-DNA sample was diluted 2000-fold.

[0050] Figure 5 This is a schematic diagram of the sensitivity electrophoresis results of multiple PCR for common Thrips, M-100bp marker, 1-single-end sample DNA without dilution, 10-DNA sample 10-fold dilution, 100-DNA sample 100-fold dilution, 500-DNA sample 500-fold dilution,

[0051] The 1000-DNA sample was diluted 1000-fold, and the 2000-DNA sample was diluted 2000-fold. DETAILED DESCRIPTION

[0052] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0053] The purpose of the present invention is to provide a multiplex PCR primer and method for quickly identifying three species of Thripidae insects. First, the present invention successfully takes the COI gene difference partial sequence region of three Thripidae insects, namely, Thrips tabacum, Thrips rubrum, and Thrips rubrum, as the research object, takes into account the difference in the length of the PCR product, respectively designs a pair of COI gene fragment amplification primers common to the three Thrips, and also designs specific primers with specific amplification for the three Thripidae insects, that is, a total of 4 pairs (8) of amplification primers are designed. The present invention optimizes its reaction system by continuously changing the multiplex polymerase chain reaction conditions such as the overall dosage of the screening reagent and the annealing temperature, successfully screens out the best annealing temperature of the specific primer, and greatly avoids the probability of forming primer dimers between primers due to the increase in the number of primers. At the same time, the present invention ensures that only one multiplex PCR reaction is needed to quickly and accurately identify the monomers of the three Thripidae insects, namely, the mixed population genomic DNA samples. Subsequent experiments have shown that accurate identification results can be obtained for DNA samples of both single thrips and mixed populations.

[0054] Specific experimental data are shown in the examples below.

[0055] Example 1 Extraction of genomic DNA from monomers and different mixed populations of three Thripidae insects

[0056] 1. Experimental Materials

[0057] Experimental samples of three known Thripidae insects, including tobacco thrips, comb thrips and common thrips.

[0058] 2. Test methods

[0059] 1) Before the genome preparation of the three known species of Thripidae insect experimental samples, the insect bodies were first soaked in 75% ethanol and wiped to remove exogenous contamination. After the ethanol evaporated, the insect bodies were frozen with liquid nitrogen, and the insect bodies were fully ground as genome extraction samples. The three known species of Thripidae insect monomer genomic DNA were extracted using a DNA extraction kit (Ezup Column Animal GenomicDNA Purification Kit, Shanghai Sangon Biotechnology Co., Ltd.), and the concentration of the extracted template DNA was detected using a spectrophotometer, and the sample DNA concentration was diluted to 20 ng / μL using the CE buffer in the DNA extraction kit, and the DNA was kept at -20°C.

[0060] 2) Extraction of genomic DNA from different mixed populations of three Thripidae insects: Take one head of each known species of Thripidae insects and mix them according to the experimental design (the mixed combinations are: tobacco thrips, comb flower thrips and common giant thrips). Use DNA extraction kit (Ezup Column Animal Genomic DNA Purification Kit, Shanghai Sangon Biotechnology Co., Ltd.) to extract genomic DNA of the above mixed population samples, use a spectrophotometer to detect the concentration of the extracted template DNA, and use the CE buffer in the DNA extraction kit to dilute the sample DNA concentration to 20 ng / μL, and keep the DNA at -20℃.

[0061] Example 2 PCR system and procedure

[0062] 1. Primer design

[0063] In this embodiment, the difference in the COI gene sequence region of three Thripidae insects, Thrips tabaci, Frankliniella schultzeiTrybom, and Megalurrothrips usitatus, was used as the research object, and the difference in the length of the PCR product was taken into account. A pair of COI gene fragment amplification primers common to the three thrips were designed respectively. At the same time, based on the designed pair of COI gene fragment amplification primers common to the three thrips, three pairs of specific primers with specific amplification for the three Thripidae insects were designed, that is, a total of 4 pairs (8) of amplification primers were designed.

[0064] The specific sequences are shown in Table 1.

[0065] Table 1 Primer sequence information

[0066]

[0067]

[0068] 2. Reaction system

[0069] 1) The multiplex PCR reaction system for the genomic DNA of three monomeric Thripidae insects and three mixed Thripidae insect populations was: 2×Gold Multiplex PCR Mix (Kangwei Century Biotechnology Co., Ltd.) 10 μL; three pairs of thrips-specific primers, a total of six, the primer concentration was 10 μM, the dosage was 0.4 μL; DNA template 1 μL; finally, sterile ultrapure water 6.6 μL was added to the reaction system to make up the reaction system to 20 μL.

[0070] 2) The reaction conditions for multiplex PCR amplification were as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 56°C for 1 min, and extension at 72°C for 30 s, for a total of 35 cycles; and extension at 72°C for 8 min.

[0071] Example 3 Specific detection of three species of thrips by multiplex PCR system

[0072] 1. Experimental methods

[0073] Tobacco thrips, comb flower thrips, common giant thrips, pipe thrips, apple thrips, and palm thrips were selected as test samples, and the genome was extracted according to the method of Example 1. According to the reaction conditions of Example 2, PCR detection was performed using single primers and mixed primers, respectively.

[0074] 2. Experimental results

[0075] The multiplex PCR products were detected by 2% agarose gel electrophoresis, and 100 bp DNA Ladder was used as a reference to determine the size of the PCR products, so as to identify the species of the Thripidae insect monomers and groups to be tested.

[0076] in, Figure 1 The results of three pairs of single primers, SEQ ID NO: 3-4, SEQ ID NO: 5-6, and SEQ ID NO: 7-8, were used to detect single samples of thrips. Figure 2 The results are SEQ ID NOs: 3 to 8 for the detection of multiple thrips samples, wherein: Thrips tabaci is 104 bp, Thrips comfrey is 237 bp, and Thrips spp. is 439 bp.

[0077] from Figure 1 The results show that SEQ ID NO: 3-4 has a detection effect only on Thrips tabaci, SEQ ID NO: 5-6 has a detection effect only on Thrips oleraceus, and SEQ ID NO: 7-8 has a detection effect only on Thrips oleraceus. The three pairs of specific primers found in the present invention have good specificity. Figure 2 The results show that the mixed primer combination also has excellent detection specificity.

[0078] At the same time, in order to further verify the reliability of the identification method, the PCR target bands corresponding to the three thrips were sent to Qingke Biotechnology Co., Ltd. (Haikou) for sequencing. The sequences of the DNA fragments obtained by sequencing were compared with the three existing sequences of Thripidae insects in NCBI, showing that both had a very high homology (more than 97%), thus confirming the reliability and accuracy of the method.

[0079] Example 4 Determination of the minimum detection limit of Thrips tabaci by multiplex PCR system

[0080] (1) The tobacco thrips DNA obtained in Example 1 was diluted with CE buffer at 1, 10, 100, 500, 1000, and 2000 times, and 1 μL was taken from each concentration as a DNA template to amplify the DNA template. The reaction system was: 2×Gold Multiplex PCR Mix (Kangwei Century Biotechnology Co., Ltd.) 10 μL; three pairs of thrips specific primers, a total of six, the primer concentration was 10 μM, and the amount was 0.4 μL; DNA template 1 μL; finally, 6.6 ul of sterile ultrapure water was added to the reaction system to make the reaction system complete to 20 μL. The PCR reaction conditions were: 95°C pre-denaturation for 10 min; 95°C deformation for 30 s, 56°C annealing for 1 min, 72°C extension for 30 s, 72°C extension for 8 min, and a total of 35 cycles.

[0081] (2) The PCR product was sequenced and the concentration was tested to calculate the copy number. The PCR amplification product was analyzed and judged using 2% gel electrophoresis. Figure 3 As shown, M-100bp marker, 1-single-end sample DNA without dilution, 10-DNA sample 10-fold dilution, 100-DNA sample 100-fold dilution, 500-DNA sample 500-fold dilution, 1000-DNA sample 1000-fold dilution, 2000-DNA sample 2000-fold dilution. The detection limit of Thrips tabaci DNA is 500 copies.

[0082] Example 5 Determination of the minimum detection limit of Thrips punctatus by multiplex PCR system

[0083] (1) The DNA of Thrips spp. obtained in Example 1 was diluted with CE buffer at 1, 10, 100, 500, 1000, and 2000 times, and 1 μL was taken from each concentration as a DNA template to amplify the DNA template. The reaction system was: 10 μL of 2×Gold Multiplex PCR Mix (Kangwei Century Biotechnology Co., Ltd.); six pairs of specific primers for Thrips, each with 0.4 μL; 1 μL of DNA template; finally, 6.6 ul of sterile ultrapure water was added to the reaction system to make the reaction system complete to 20 μL. The PCR reaction conditions were: 95°C pre-denaturation for 10 min; 95°C deformation for 30 s, 56°C annealing for 1 min, 72°C extension for 30 s, 72°C extension for 8 min, for a total of 35 cycles.

[0084] (2) The PCR product was sequenced and the concentration was tested to calculate the copy number. The PCR amplification product was analyzed and judged using 2% gel electrophoresis. Figure 3As shown, M-100bp marker, 1-single-end sample DNA without dilution, 10-DNA sample 10-fold dilution, 100-DNA sample 100-fold dilution, 500-DNA sample 500-fold dilution, 1000-DNA sample 1000-fold dilution, 2000-DNA sample 2000-fold dilution. The detection limit of DNA of Thrips spp. is 100 copies.

[0085] Example 6 Determination of the minimum detection limit of common Macrothrips by multiplex PCR system

[0086] (1) The DNA of common giant thrips obtained in Example 1 was diluted with CE buffer at 1, 10, 100, 500, 1000, and 2000 times, and 1 μL was taken from each concentration as a DNA template to amplify the DNA template. The reaction system was: 10 μL of 2×Gold Multiplex PCR Mix (Kangwei Century Biotechnology Co., Ltd.); six pairs of specific primers for thrips, each with 0.4 μL; 1 μL of DNA template; finally, 6.6 ul of sterile ultrapure water was added to the reaction system to make the reaction system complete to 20 μL. The PCR reaction conditions were: 95°C pre-denaturation for 10 min; 95°C deformation for 30 s, 56°C annealing for 1 min, 72°C extension for 30 s, 72°C extension for 8 min, and 35 cycles in total.

[0087] (2) The PCR product was sequenced and the concentration was tested to calculate the copy number. The PCR amplification product was analyzed and judged using 2% gel electrophoresis. Figure 4 As shown, M-100bp marker, 1-single-end sample DNA without dilution, 10-DNA sample 10-fold dilution, 100-DNA sample 100-fold dilution, 500-DNA sample 500-fold dilution, 1000-DNA sample 1000-fold dilution, 2000-DNA sample 2000-fold dilution. The detection limit of common Thrips DNA is 100 copies.

[0088] Based on the results of Examples 4, 5, and 6, the detection limits of the mixed detection primers obtained in the present invention for different thrips are shown in Table 2.

[0089] Table 2 Detection limits of fragment sizes and DNA concentrations corresponding to target species

[0090]

[0091] In summary, the identification primers and multiplex PCR system of the present invention can quickly, effectively and accurately identify these three thrips, and the PCR product band sizes of tobacco thrips, comb-deficient flower thrips and common large thrips are 104bp, 237bp and 439bp respectively. The detection limit of sensitivity tobacco thrips is 500 copies, and the detection limits of comb-deficient flower thrips and common large thrips are both 100 copies. Compared with ordinary PCR, which can only identify one type of thrips at a time, the multiplex PCR system can complete the identification of multiple types of thrips at a time, and has the advantages of simple operation, low cost, and time saving.

Claims

1. A multiplex PCR detection primer for detecting thrips, characterized in that: The thrips include Thrips tabaci, Thrips oleraceus and Thrips spp. The multiplex PCR detection primers include specific primers for detecting Thrips tabaci, Thrips oleraceus and Thrips spp.; The specific primer sequences for detecting Thrips tabaci are shown in SEQ ID NOs: 3 and 4; The specific primer sequences for detecting thrips are shown in SEQ ID NOs: 5 and 6; The specific primer sequences for detecting common giant thrips are shown in SEQ ID NOs: 7 and 8.

2. Use of the multiplex PCR detection primers according to claim 1 in at least one of (a1) to (a6): (a1) Identification of Thrips tabaci; (a2) preparing products for identifying Thrips tabaci; (a3) Identification of thrips; (a4) preparing a product for identifying Thrips spp.; (a5) Identification of common Macrothrips; (a6) Preparation of a product for identifying common Thrips.

3. The use according to claim 2, characterized in that: The product includes at least one of a reagent, a test kit, a test paper, a chip, and a system.

4. A kit for detecting thrips, characterized in that: The kit comprises the multiplex PCR detection primers according to claim 1.

5. The kit according to claim 4, characterized in that: The kit also includes Taq DNA polymerase, dNTP, PCR buffer, Mg 2+ At least one of .

6. A method for identifying thrips, characterized in that: The following steps are involved: 1) using the multiplex PCR detection primers described in claim 1 or the kit described in claim 4 or 5 to perform PCR amplification on the sample to be tested; 2) Analyze the size of the amplified product and identify the sample type based on the size of the amplified product band.

7. The method according to claim 6, characterized in that: The reaction conditions of the PCR amplification are as follows: based on a 20 μL reaction system, it includes 10-100 ng of DNA template, 0.1-0.3 μM of multiple PCR primer concentration, 10 μL of 2×PCR Buffer, and the rest is ddH2O.

8. The method according to claim 6, characterized in that: Pre-deformation at 95℃ for 10min; deformation at 95℃ for 30s; annealing at 56℃ for 1min, extension at 72℃ for 30s, for a total of 35 cycles; extension at 72℃ for 8min.

9. The method according to claim 6, characterized in that: The thrips include Thrips tabaci, Thrips oleraceus and Thrips spp.

10. The method according to claim 9, characterized in that: When the amplified product showed a 104 bp band, it was determined that the sample contained Thrips tabaci; When the amplified product has a 237 bp band, it is judged that the sample contains Thrips punctatus; When a 439 bp band is present in the amplified product, it is determined that the sample contains Macrothrips cyprinidae.

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