Specific primer and method for rapidly identifying leaf miner

Abstract

The invention discloses specific primers and a method for rapidly identifying leaf miners, and belongs to the technical field of molecular biology. According to the method, rapid identification of leaf miners is realized mainly by designing specific primers. The primer disclosed by the invention has high specificity, only performs specific amplification on the COI gene of the leaf miner, and does not have an amplification product on sibling species such as Lithocolletis ringoniella, Grapholitha molesta, Ampelopsis molesta, codling moth and Phytophorus nigricans. The primer is suitable for detecting different development stages (first-instar larvae, second-instar larvae, third-instar larvae, pupae and adults) of leaf miners and different tissue parts (tentacles, head chest, abdomen, wings and feet) of the adults, the annealing temperature range is wide, and the sensitivity is high. The method is easy and convenient to operate, the detection time is short (2-3 hours), the method can be widely applied to port quarantine, field monitoring and other scenes, and technical support is provided for precise prevention and control of leaf miners.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology. Specifically, it relates to a specific primer and method for rapid identification of the leafminer moth, which is particularly suitable for the accurate identification of the leafminer moth and its closely related species in plant quarantine, port quarantine and field monitoring. Background Technology

[0002] The spiral leafminer is a globally distributed and important leaf-mining pest of fruit trees, belonging to the family Lyonetiidae in the order Lepidoptera. Native to Europe, this insect is now widely distributed in temperate regions of Eurasia and continues to expand to other fruit-producing areas worldwide. Recently, it was first recorded in Tunisia, North Africa, raising serious concerns. Its host range is mainly concentrated on Rosaceae fruit trees, including apples, pears, crabapples, crabapples, and hawthorns. The larvae burrow between the leaf epidermis and feed on the leaf tissue, forming typical concentric spiral-shaped spots. In severe cases, this leads to premature leaf drop, weakened tree vigor, and a significant decrease in fruit yield and quality, causing direct economic losses to orchards.

[0003] Morphological characteristics: Adults are small, 2-3 mm in body length and 6-8 mm in wingspan. The body and legs are silvery-white, with erect white tufts of hair on the crown. The basal half of the forewing is white, with irregular orange-yellow spots near the tip, often edged with brown. The anal region has black spots and silvery-white and purplish-black scales on the inner side; the fringe is white with several dark brown transverse bands. Eggs are flat and oval, about 0.3 mm long, with reticulate ridges on the surface. Initially laid, they are light green or grayish-white, translucent and glossy. Mature larvae are 4-5.5 mm long, yellowish-white with a slight green tinge, with a black head. The pronotum (and sternum) has a large rectangular black spot in the center. The mesothorax and abdominal segments 1-2 have nipple-like projections on both sides, with a fine hair at the tip. Pupae are 3-5 mm long, flat and spindle-shaped, light brown to dark brown. Before pupation, the larva spins an "H"-shaped silk ribbon on the substrate surface, then forms a thin cocoon beneath the ribbon and pupates inside.

[0004] Against the backdrop of global warming and increasingly frequent international agricultural trade, the risk of long-distance spread of the leafminer moth via seedlings, fruits, and transportation vehicles has increased dramatically. Due to its tiny size and the striking similarities in wing markings, larval morphology, and damage among several closely related species, including common orchard moths, traditional morphological identification relies heavily on complete adult specimens and specialized lepidopteran and genital anatomy comparison techniques. This process is time-consuming and demands a high level of experience from the identification personnel. In practical scenarios such as port quarantine and field monitoring, samples are often incomplete forms, such as eggs, young larvae, pupae, broken adults, or molted skin. This often renders traditional morphological identification methods ineffective, easily leading to misjudgments or missed detections, thereby increasing the risk of quarantine loopholes and delays in prevention and control.

[0005] To address this challenge, molecular biology techniques have become crucial tools for precise species identification. Among these, mitochondrial DNA (mtDNA), due to its simple structure, rapid evolutionary rate, maternally inherited nature, relative conservation within species, and significant interspecific differences, is widely used for phylogenetic analysis and rapid identification primer development in insect relatives and subspecies. In particular, the cytochrome C oxidase subunit I (COI) gene, as a standard "DNA barcode" region, has demonstrated high effectiveness and reliability in species identification of Lepidoptera insects.

[0006] Although some studies have used molecular markers to identify some leafminer moths, existing technologies still have significant limitations: on the one hand, most studies lack systematic primer design and validation for the spiral leafminer and its common closely related species, resulting in insufficient primer specificity and the possibility of false positive or false negative results in practical applications; on the other hand, existing methods have limited sensitivity when detecting different insect developmental stages (especially trace DNA samples such as first-instar and second-instar larvae), and PCR reaction conditions (such as annealing temperature) are often demanding and the operation process is complex, making it difficult to meet the needs of rapid and high-throughput detection at ports of entry.

[0007] Therefore, developing a rapid molecular identification technique that is highly specific, sensitive, easy to operate, and applicable to whole-life and fragmented samples of the leafminer moth has become an urgent need for current plant quarantine and integrated pest management. Summary of the Invention

[0008] The purpose of this invention is to provide specific primers and methods for rapid identification of the leafminer moth. This method achieves rapid identification of the leafminer moth by designing specific primer pairs. This method is not limited by different tissues and optimizes the primers and amplification system for rapid identification of the leafminer moth and closely related species, providing a rapid and accurate method for identifying quarantine pests in import and export and for plant quarantine work.

[0009] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a specific primer for rapid identification of the leafminer moth, the primer consisting of a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2.

[0010] Specifically, SEQ ID NO.1 is: CCTGCTAATACTGGTAATGA.

[0011] SEQ ID NO.2 is specifically: CAGGTATAATTGGAACATCC.

[0012] Furthermore, the primers can specifically amplify the COI gene in the mitochondrial DNA genome of the leafminer moth, but cannot amplify the five closely related species of the leafminer moth; the five closely related species of the leafminer moth are the golden leafminer moth, the pear fruit moth, the apple leafroller moth, the codling moth, and the black leafroller moth.

[0013] Furthermore, the nucleotide sequence of the COI gene is shown in SEQ ID NO.3, specifically as follows:

[0014] The present invention also provides the application of the specific primers in the preparation of a kit for identifying the leafminer moth.

[0015] This method also provides a kit for rapid identification of the leafminer moth, the kit containing the specific primers.

[0016] The present invention also provides a method for identifying the leafminer moth using the specific primers, comprising the following steps: S1. Extract genomic DNA from the sample to be tested; S2. Using the DNA extracted in step S1 as a template, perform PCR amplification using the specific primers. S3. Perform agarose gel electrophoresis analysis on the PCR amplification products; S4. If the electrophoresis results show the amplification of specific bands, the sample is identified as a leaf miner moth.

[0017] Furthermore, the PCR amplification reaction system is as follows: 12.5 μL of 2× Taq PCR mix, 1 μL each of forward and reverse primers, 1 μL of DNA template, 9.5 μL of sterile distilled water, and a total volume of 25 μL.

[0018] Furthermore, the annealing temperature in the PCR amplification reaction program is 50℃-60℃.

[0019] Furthermore, the method is applicable to different developmental stages of the leafminer moth, including first instar larvae, second instar larvae, third instar larvae, pupae, and adults.

[0020] Furthermore, the method is applicable to different tissue parts of adult leafminer moths, including antennae, head and thorax, abdomen, wings and legs.

[0021] The beneficial effects of this invention are: (1) The primers designed in this invention are highly specific, and only amplify bands for the leafminer moth, while not amplifying bands for closely related species such as the golden leafminer moth, pear fruit moth, apple leafroller moth, codling moth, and black leafroller moth.

[0022] (2) The present invention has a wide range of applications, and has detected every instar and developmental stage from larva to adult, proving that the primer is capable of detecting each stage of the leafminer moth.

[0023] (3) The PCR system of the present invention is simple and convenient. It only requires the addition of primers and templates to carry out amplification, thus optimizing the amplification system.

[0024] (4) The primers provided by this invention have a wide annealing temperature range, and the target species can be accurately detected within the range of 50℃ to 60℃ without producing false positives.

[0025] (5) The present invention takes into account the situation where multiple moth tissues are mixed and cannot be identified during the quarantine process, and proposes mixed DNA detection of quarantine species. After testing, the primers of the present invention have sufficiently high sensitivity to detect low concentration templates of the leaf miner moth. Attached Figure Description

[0026] Figure 1 Electrophoresis images show the amplification of five common apple orchard species of the leafminer moth and its close relatives using primer pairs with nucleotide sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5. M: DL2000 DNA marker (2000, 1000, 750, 500, 250, 100 bp from top to bottom); Lane 1 represents the leafminer moth (…). Leucoptera malifoliella Lane 2 is for the golden-striped fine moth ( Lithocolletis ringoniella Lane 3 is for pear fruit moth ( ), Grapholita molesta Lane 4 is for the apple leafroller moth ( Adoxophyes orana Lane 5 is inhabited by codling moths ( Cydia pomonella Lane 6 is for the plant black leafroller ( ), Endothenia oblongana ).

[0027] Figure 2 Electrophoresis images show the amplification of five common apple orchard species of the leafminer moth and its close relatives using primer pairs with nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2. M: DL2000 DNA marker (2000, 1000, 750, 500, 250, 100 bp from top to bottom); Lane 1 represents the leafminer moth (…). L. malifoliella Lane 2 is for the golden-striped fine moth ( L. ringoniella Lane 3 is for pear fruit moth ( ), G. molesta Lane 4 is for the apple leafroller moth ( A. orana Lane 5 is inhabited by codling moths ( C. pomonella Lane 6 is for the plant black leafroller ( ), E. oblongana ).

[0028] Figure 3 The image shows the results of determining the optimal annealing temperature for primer pairs with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2. In the image, M represents the DL2000 DNA marker; lanes 1-8 represent different annealing temperatures, namely 60℃, 59℃, 58℃, 56℃, 55℃, 54℃, 52℃, and 50℃, respectively.

[0029] Figure 4 This diagram shows the results of testing the universality of primer pairs shown in SEQ ID NO.1 and SEQ ID NO.2 using tissue DNA from different developmental stages of the leafminer moth as templates. M represents the DL2000 DNA marker; lanes 1-5 represent the first instar, second instar, third instar, pupa, and adult stages of the leafminer moth, respectively.

[0030] Figure 5 This diagram shows the results of testing the universality of primer pairs shown in SEQ ID NO.1 and SEQ ID NO.2 using DNA from different tissues of the leafminer moth as templates. M represents the DL2000 DNA marker; lanes 1-5 represent the antennae, cephalothorax, abdomen, wings, and legs of the leafminer moth, respectively.

[0031] Figure 6The graph shows the sensitivity test results of the primer pairs with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2. M: DL2000 DNA marker; lanes 1-6 represent undiluted, 10-fold, 100-fold, 1000-fold, 10000-fold, and 100000-fold dilutions, respectively. Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments. Example

[0033] 1. Extraction of insect genomic DNA Genomic DNA was extracted from specimens of the leafminer moth and closely related species using the OMEGA Genomic DNA Miniature Kit. The specific procedures are as follows: (1) Pulverize no more than 50 mg of insect tissue (prothorax or leg) in liquid nitrogen using a mortar and pestle, and transfer the powder to a clean 1.5 mL microcentrifuge tube.

[0034] (2) Add 350 μl Buffer CTL and 25 μl Proteinase K, vortex and mix well, then incubate at 60 °C for 30 min until the sample is completely dissolved.

[0035] (3) Add 350 μl of chloroform:isoamyl alcohol (24:1) mixture, vortex to mix, centrifuge at 10,000 x g for 5 min at room temperature, and carefully transfer the supernatant to a new 1.5 mL centrifuge tube.

[0036] (4) Add an equal volume of Buffer CBL, vortex at maximum speed for 15s, and incubate at 60℃ for 10min.

[0037] (5) Add an equal volume of anhydrous ethanol and vortex mix at maximum speed for 15s.

[0038] (6) Insert the HiBind® DNA column into a 2mL collection tube, add 750μl of the mixture from step 5, centrifuge at 10,000xg for 1 min at room temperature, and discard the filtrate.

[0039] (7) Put the HiBind® DNA column back into the 2mL collection tube, add 500μl Buffer HB, centrifuge at 10,000xg for 1min, and discard the filtrate.

[0040] (8) Insert the HiBind® DNA column into a new 2mL collection tube, add 700μl DNA WashBuffer, centrifuge at 10,000xg for 1min, discard the filtrate and repeat the operation once.

[0041] (9) Put the HiBind® DNA column back into the 2mL collection tube, and centrifuge the empty column at 10,000xg for 2 min at room temperature to dry.

[0042] (10) Insert the HiBind® DNA column into a new 1.5mL centrifuge tube, add 50-100μl of preheated Elution Buffer at 60℃, let stand at room temperature for 2min, centrifuge at 10,000xg for 1min to elute the DNA.

[0043] (11) DNA concentration detection. The concentration of extracted DNA was detected using an ultra-micro spectrophotometer. Before detection, the detection wells were cleaned with deionized water, dried, and then 1 μL of the elution buffer used during DNA extraction was applied for correction. After correction, 1 μL of sample was applied to the detection wells, and the concentration of all DNA samples was measured.

[0044] 2. Amplification of target genes in the leafminer moth and its close relatives The COI gene from the mitochondrial DNA genome was selected for the design of specific primers. A pair of universal primers suitable for the modified COI gene of the mitochondria in the lepidopteran silkworm, as described in the literature, was synthesized. After sequence alignment, degenerate primers were synthesized to amplify the mitochondrial COI gene of six species. The universal primer sequences are as follows: LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3' (SEQ ID NO.4); HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (SEQ ID NO. 5).

[0045] The total PCR reaction volume was 25 μL, including 12.5 μL of 2× Taq PCR mix, 1 μL each of the upstream primer (SEQ ID NO. 4) and the downstream primer (SEQ ID NO. 5), 1 μL of DNA template, and then adding sterile distilled water to a final volume of 25 μL. The reaction conditions were as follows: 94°C pre-denaturation for 3 minutes, 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 30 cycles, followed by a final extension at 72°C for 1 minute and a final extension at 72°C for 5 minutes. The product was stored at 4°C. Subsequently, the PCR product (5 μL) was analyzed by 1.5% agarose gel electrophoresis. Non-toxic gelred dye was added during agarose gel preparation, and fragment sizes were labeled using DL2000 DNA marker. Agarose gel electrophoresis showed that bright bands were amplified in all six lepidopteran moth species. Figure 1 The banded samples were sent to Beijing Qingke Company for bidirectional sequencing.

[0046] 3. SS-COI primer design for the leafminer moth Sequence alignment of COI fragments from six species of the leafminer moth and common apple orchard relatives was performed using Bioedit software. Conserved but significantly different fragments within the leafminer moth and its relatives were identified for specific primer design. In this study, a pair of specific primers for the leafminer moth, SXW(F) and SXW(R), were designed using Primer 5.0. The primer sequences are as follows: SXW(F): 5'-CCTGCTAATACTGGTAATGA-3' (forward primer, SEQ ID NO.1); SXW(R): 5'-CAGGTATAATTGGAACATCC-3' (reverse primer, SEQ ID NO.2).

[0047] 4. Specificity test of SS-COI primers for the leafminer moth (1) Five samples were selected for each species for testing. The PCR reaction system was 25 μL, including 12.5 μL of 2×taqPCR mix, 1 μL each of the forward primer (SEQ ID NO.1) and the reverse primer (SEQ ID NO.2), 1 μL of DNA template, and then sterile distilled water was added to 25 μL. The reaction program was 94℃ pre-denaturation for 3 minutes, 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 30 cycles, followed by extension at 72℃ for 1 minute, and finally extension at 72℃ for 5 minutes. Agarose gel electrophoresis showed that only the leafminer moth could amplify a bright band ( Figure 2 (), while none of the five closely related species of the spiral leafminer amplified the bands.

[0048] (2) Eight annealing temperatures were set for temperature gradient PCR experiments to explore the optimal annealing temperature, ranging from 50℃ to 60℃. Electrophoresis results showed that the specific primers for the leafminer moth designed in this invention exhibited a wide range of annealing temperatures, producing the target band for the leafminer moth from 50℃ to 60℃. Figure 3 This indicates that the primer pair has good stability and specificity, and has amplification effect on samples at multiple annealing temperatures, which can rule out inaccurate results caused by temperature differences between different PCR instruments.

[0049] (3) Genomic DNA was extracted from first-instar larvae, second-instar larvae, third-instar larvae, pupae, and adults of the leafminer moth and used as templates. Specific primer pairs (SEQ ID NO.1, SEQ ID NO.2) were used for detection. According to the electrophoresis results, these primer pairs amplified the target bands for different developmental stages and instars of the leafminer moth. Figure 4 ).

[0050] (4) The adult leafminer was divided into five parts based on common remains: antennae, head and thorax, abdomen, wings, and legs. Genomic DNA was extracted from each part and used as templates. Specific primer pairs (SEQ ID NO.1, SEQ ID NO.2) were used for detection. According to the electrophoresis results, the primer pairs amplified the target bands in different parts of the adult leafminer. Figure 5 ).

[0051] 5. Sensitivity test of specific primers for the leafminer moth The leafminer moth template was diluted with sterile water at ratios of (1:1), (1:10), (1:100), (1:1000), (1:1000), (1:10000), and (1:100000) to test the sensitivity of specific primers. The concentration of the leafminer moth standard solution was 30 ng / µl. Electrophoresis results showed that the target band appeared at all concentration gradients except for 1:10000 and 1:100000. Figure 6 ).

[0052] In summary, this invention achieves rapid identification of the leafminer moth by designing specific primers (SEQ ID NO.1, SEQ ID NO.2). The primers designed in this invention are highly specific, amplifying bands only for the leafminer moth, while failing to amplify bands for closely related species such as the golden leafminer moth, pear fruit moth, apple leafroller, codling moth, and black leafroller. These primers are suitable for detecting different developmental stages of the leafminer moth (first instar larvae, second instar larvae, third instar larvae, pupa, and adult) and different tissue parts of the adult (antennae, head, thorax, abdomen, wings, and legs). They have a wide annealing temperature range (50℃-60℃) and high sensitivity (detecting DNA templates at a dilution of 1:1000).

Claims

1. A specific primer for rapid identification of the leafminer moth, characterized in that, The primers consist of a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.

2.

2. The specific primer according to claim 1, characterized in that, The primers can specifically amplify the COI gene in the mitochondrial DNA genome of the leafminer moth, but cannot amplify the five closely related species of the leafminer moth; the five closely related species of the leafminer moth are the golden leafminer moth, the pear fruit moth, the apple leafroller moth, the codling moth, and the black leafroller moth.

3. The specific primer according to claim 2, characterized in that, The nucleotide sequence of the COI gene is shown in SEQ ID NO.

3.

4. The use of the specific primers described in claim 1 in the preparation of a kit for identifying the leafminer moth.

5. A kit for rapid identification of the leafminer moth, characterized in that, The kit contains the specific primers as described in claim 1.

6. A method for identifying the leafminer moth using the specific primers described in claim 1, characterized in that, Includes the following steps: S1. Extract genomic DNA from the sample to be tested; S2. Using the DNA extracted in step S1 as a template, perform PCR amplification using the specific primers described in claim 1; S3. Perform agarose gel electrophoresis analysis on the PCR amplification products; S4. If the electrophoresis results show the amplification of specific bands, the sample is identified as a leaf miner moth.

7. The method according to claim 6, characterized in that, The PCR amplification reaction system consisted of 12.5 μL of 2×taqPCR mix, 1 μL each of forward and reverse primers, 1 μL of DNA template, 9.5 μL of sterile distilled water, and a total volume of 25 μL.

8. The method according to claim 6, characterized in that, The annealing temperature in the PCR amplification reaction program is 50℃-60℃.

9. The method according to claim 6, characterized in that, The method is applicable to different developmental stages of the leafminer moth, including first instar larvae, second instar larvae, third instar larvae, pupae, and adults.

10. The method according to claim 6, characterized in that, The method is applicable to different tissue parts of adult leafminer moths, including antennae, head and thorax, abdomen, wings and legs.

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