A primer set, kit and method for detecting tomato leafminer

Abstract

This invention relates to the field of biological detection technology, specifically to a method for detecting tomato leafminer based on recombinase-mediated isothermal nucleic acid amplification (RAA) technology. Tuta absoluta = Phthorimaea absoluta Animal mitochondria COI This invention discloses a primer set, kit, and method for detecting tomato leafminer. The forward primer sequence for detecting tomato leafminer designed in this invention is shown in SEQ ID No. 1, the reverse primer sequence is shown in SEQ ID No. 2, and the probe primer for detecting tomato leafminer is shown in SEQ ID No. 3. The primer set of this invention can effectively amplify a portion of the tomato leafminer. COⅠ and COⅡ The gene and primer set have high specificity and sensitivity, and show no cross-reactivity with other leafminer insects that coexist with the tomato leafminer. When used in conjunction with lateral flow immunochromatography, it can be used for rapid on-site identification of the tomato leafminer at various stages, which is of great significance for the effective prevention and control of the tomato leafminer.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a primer set, kit, and method for detecting the COI barcode of mitochondria in the tomato leafminer moth based on recombinase-mediated isothermal nucleic acid amplification (RAA) technology. Background Technology

[0002] The tomato leafminer (Tuta absoluta) (Meyrick), also known as the tomato leafminer moth, is a major pest of the Lepidoptera family Meciidae, often referred to as the "Ebola virus of tomatoes." Originating in South America, it was first discovered and described in Huancayo, Peru in 1917. It began to spread throughout South America in the mid-20th century. It has a wide host range, damaging 40 species of crops from 19 families, and in severe outbreaks can cause 80%–100% of tomato yield loss.

[0003] The tomato leafminer spreads rapidly. First discovered in Xinjiang, my country in 2017, it has now spread to Northwest, Southwest, North, Central, and Northeast my country, causing localized outbreaks. The tomato leafminer has a wide host range, with 50 species belonging to 11 families, including fruits and vegetables such as tomatoes, watermelons, ginseng fruit, ground cherries, eggplants, peppers, cucumbers, and beans; grain and vegetable crops (such as potatoes); oil crops (such as soybeans); cash crops (such as goji berries, tobacco, sugar beets, and alfalfa); and the globally widespread weed black nightshade. In 2023, the tomato leafminer was included in the "List of Class A Crop Diseases and Pests" by the Ministry of Agriculture and Rural Affairs of China.

[0004] The tomato leafminer primarily damages the plant as larvae, which can infest any stage of tomato plant development and any above-ground part. They feed on leaf tissue, fruit, terminal buds, and tender shoots and stems. The larvae initially create tiny tunnels within the leaf tissue, which are usually difficult to detect in the early stages due to their excellent concealment. As the larvae grow and their appetite increases, the tunnels widen and enlarge, forming irregular, translucent spots, eventually causing the affected leaves to wrinkle and dry out. Older larvae can also bore into terminal shoots, axillary buds, tender stems, and young fruit.

[0005] The tomato leafminer is small, with adults measuring 6-7 mm in length, while eggs and newly hatched larvae are less than 1 mm long. Tomato leafminer eggs are approximately 0.36 mm long and 0.22 mm wide, cylindrical in shape, ranging from milky white to orange-yellow, and are difficult to spot with the naked eye. Females lay eggs on the undersides and tops of leaves, buds, stems, or green fruits. Once hatched, the larvae burrow into leaves, stems, or fruits to feed. While burrowing, the larvae are difficult to completely separate from the plant leaves, making accurate field detection of trace samples such as tomato leafminer eggs and larvae extremely challenging. The tomato leafminer feeds by burrowing or boring, making it difficult to detect in a timely manner. Its long-distance dispersal is mainly through trade / transportation of agricultural products (especially tomatoes and seedlings), as well as through transport vehicles, containers, fillers, and packaging materials. Short- and medium-distance dispersal relies primarily on natural factors or its own capabilities, such as air currents, but its ability to fly independently requires further research and confirmation. Furthermore, the role of the sale / purchase of infected solanaceous vegetables, especially tomato seedlings, in their spread cannot be ignored.

[0006] Currently, countries around the world are taking measures to block the spread of the tomato leafminer. The main methods for detecting and identifying the tomato leafminer are nucleic acid-based molecular biological assays (PCR and LAMP, etc.), with PCR being the most commonly used. Because species identification often uses mitochondrial genomic DNA as a template for amplification and involves DNA extraction procedures, this process is crucial.

[0007] RT-RAA (reverse transcription recombinase polymerase amplification) is an emerging molecular biology technique that utilizes recombinases obtained from bacteria or fungi. Reverse transcriptase and recombinase bind tightly to primer DNA, forming an enzyme-primer polymer. When the primers find a perfectly matching complementary sequence on the template DNA / RNA, the double-stranded structure of the template DNA / RNA is opened with the help of single-stranded DNA-binding proteins. Under the action of reverse transcriptase and / or DNA polymerase, a new complementary DNA strand is formed, resulting in exponential growth of the amplified product. Compared to traditional PCR (polymerase chain reaction), RT-RAA offers advantages such as ease of operation, high sensitivity, rapid amplification speed, and resistance to amplification inhibitors. It can be used for the detection of DNA or RNA targets and has broad application potential in pathogen detection, SNP genotyping, and other fields.

[0008] RT-RAA reactions are performed isothermally between 37°C and 42°C, eliminating the need for expensive thermal cycling equipment. This characteristic makes RTRAA particularly suitable for on-site detection. RAA amplification products can be detected by agarose gel electrophoresis or visualized using lateral flow strips (LFS). In RT-RAA-LFS detection, the amplification products generated by the RT-RAA reaction are labeled and interact with specific probes on the LFS. These labeled products move on the LFS and form visible signals (usually lines or color changes) on the detection lines, enabling qualitative or quantitative detection of the target nucleic acid.

[0009] While molecular biology methods such as PCR and LAMP can be used for species identification of tomato leafminer mitochondrial COI barcoding, these methods still do not completely eliminate the step of nucleic acid extraction. The nucleic acid extraction and PCR detection processes either heavily rely on well-trained laboratory technicians, require complex and expensive laboratory equipment, or involve cumbersome detection procedures that are difficult to perform outside of a laboratory environment. Therefore, they cannot meet the needs for rapid and accurate field detection of suspected tomato leafminer eggs or insects. Summary of the Invention

[0010] Accurately distinguishing between the eggs or larvae on host samples and those of the tomato leafminer or other leafminer insects that coexist in the same region will directly affect the effectiveness of the selected control measures. Therefore, there is an urgent need to establish a rapid, simple, and accurate method for identifying and detecting the tomato leafminer as a specific species.

[0011] To address the aforementioned technical problems, the present invention aims to provide a primer set, kit, and method for detecting tomato leafminer based on RAA (Rapid Alternative Detection). The specific technical solution is as follows:

[0012] In one aspect, the present invention provides a species-specific RT-RAA primer set for detecting the tomato leafminer *Tuta absoluta* mitochondrial COI barcoding, the primer set comprising tomato leafminer species-specific detection primers and a detection probe Tuta-P; wherein,

[0013] The tomato leafminer detection primers include the forward primer Tuta-F and the reverse primer Tuta-R;

[0014] The sequence of the forward primer Tuta-F is: 5′-TATTTATATTAATTATTATTTGAGAA-3′ (SEQ ID NO.1), and the sequence of the reverse primer Tuta-R is: 5′-GGAGAAGTCCCATTTTGAAGATT-3′ (SEQ ID NO.2).

[0015] The mitochondrial COI barcode detection probe Tuta-P for the tomato leafminer moth has the sequence 5′-AATGATATCAATTATTACCCCCCGCAHAACATTCATATAATGAAC-3′ (SEQ ID NO.3);

[0016] In this invention, the 5′ end of the reverse primer Tuta-R is modified with FITC, the 5′ end of the detection probe Tuta-P is modified with biotin, the 3′ end of Tuta-P is phosphorylated, H is tetrahydrofuran, and all underlined bases in the Tuta-P sequence are modified with locked nucleotides.

[0017] In this invention, the RAA reaction system for detecting tomato leafminer is as follows:

[0018] Element Volume (μL) Plant sample lysate or total RNA solution 2 Basic buffer 29.4 Tuta-F (10μM) 2.1 Tuta-R (10μM) 3 Tuta-P (10μM) 0.6 <![CDATA[ddH2O]]> upto47.5

[0019] The reaction conditions for RAA amplification were set as follows: After the reaction solution was prepared, 2.5 μL of 280 mM MgAc2 was added to each 0.2 mL eppendorf tube and mixed thoroughly; the 0.2 mL eppendorf tube was placed on a PCR instrument with a heated lid and incubated at 39 °C for 16 min.

[0020] In one aspect, the present invention provides a reagent for detecting the tomato leafminer (Tuta absoluta), the reagent containing a primer set for species-specific RT-RAA detection of the mitochondrial COI barcode of the tomato leafminer (Tuta absoluta).

[0021] In one aspect, the present invention provides a kit for detecting the tomato leafminer *Tuta absoluta*, the kit containing a species-specific RT-RAA detection reagent for detecting the mitochondrial COI barcode of *Tuta absoluta*. The kit may also contain other reagents, such as DNA extraction reagents, sample lysis buffer, buffer solution, and kit instructions.

[0022] In one aspect, the present invention provides the application of the reagents and kits for detecting the tomato leafminer (Tuta absoluta) in the detection of the tomato leafminer. If the mitochondrial COI barcode sequence of the tomato leafminer (Tuta absoluta) can be detected, the biological sample is infected with the tomato leafminer.

[0023] In one embodiment of the present invention, the detection reagent or kit can be used in conjunction with a lateral flow immunochromatographic assay kit.

[0024] In one aspect, the present invention provides a method for detecting tomato leafminer based on RT-RAA-lateral flow chromatography, comprising the following steps:

[0025] 1) Perform lysis on the sample or extract genomic DNA from the biological sample to be tested;

[0026] 2) Using the sample lysate or extracted genomic DNA obtained in step 1) as a template, perform RT-RAA amplification using the primer set described in claim 1 or 2;

[0027] 3) Use lateral flow chromatography test strips to detect amplification products, observe the color development of the bands on the lateral flow chromatography test strips, and determine whether the sample contains tomato leafminer.

[0028] The method of observing the color development of the side-flow chromatography test strip to determine whether the sample contains tomato leafminer is as follows: if the test strip shows one band at the control line and no band at the test line, the test result is negative, indicating that the sample does not contain or is not in contact with tomato leafminer; if the test strip shows two bands, one at the control line and one at the test line, the result is positive, indicating that the sample contains or is in contact with tomato leafminer; if the control line of the test strip does not develop color, the test is invalid and the test strip needs to be replaced and the test repeated.

[0029] The specific detection process in this invention is as follows: Open the cap of the Eppendorf tube containing 50 μL of RTRAA amplification product, take 2.5 μL of the amplification product into a new 1.5 mL Eppendorf tube, and dilute it 100 times. Insert a new nucleic acid lateral chromatography test strip directly into the Eppendorf tube containing the diluted product, ensuring the liquid level does not exceed the upper limit mark of the sample pad immersion. After the interpretation area is completely immersed, lay the test strip flat for 1 minute, observe the color development result, and record it within 10 minutes. Each test sample should show at least one control line, with or without a test line. If the test strip shows one band at the control line and no band at the test line, the test result is negative, indicating that the sample does not contain or is not infected with tomato leafminer; if the test strip shows two bands, one at the control line and one at the test line, the result is positive, indicating that the sample contains or is infected with tomato leafminer; if the control line of the test strip does not develop color, the test is invalid, and the test strip needs to be replaced and the test repeated.

[0030] Beneficial effects

[0031] This invention provides a primer set for species-specific identification of the tomato leafminer moth, establishing a rapid field detection method for tomato leafminer eggs, larvae, pupae, and adults. The primers designed in this invention can effectively amplify the tomato leafminer mitochondrial target gene, exhibiting extremely high specificity and sensitivity. Using RAA-lateral flow chromatography, a 50 μL reaction system can detect the tomato leafminer mitochondrial target gene sequence with an initial amplified template of 1.6 pg or 51 copies. Furthermore, the detection in this invention shows no cross-reactivity with closely related species and genera of leaf-eating / leaf-mining / fruit-boring pests occurring in the same domain as the potato tuber moth. This method can be used for rapid field and on-site detection or identification of the tomato leafminer moth species, and is of great significance for plant quarantine, detection, monitoring, and control of the tomato leafminer moth. Attached Figure Description

[0032] Figure 1 RAA amplification results of genomic DNA solutions from *Tomato leafminer* and *Potato tuber moth* using the tomato leafminer-specific primers Tuta-F / Tuta-R1, Tuta-F / Tuta-R2, Tuta-F / Tuta-R, and Tuta-F / Tuta-R4, where M: molecular weight standard Trans 2K TM .

[0033] Figure 2 RAA amplification results of genomic DNA solutions of the tomato leafminer and its closely related species, leaf-eating / leaf-mining / fruit-boring pests, using the tomato leafminer-specific primers Tuta-F / Tuta-R. Where M: molecular weight standard Trans 2K. TM1. Tomato leafminer *Tuta absoluta* (Meyrick); 2. Potato tuber moth *Phthorimaea operculella* (Zeller); 3. Sweet potato leafminer *Brachmia macroscopa* (Meyrick); 4. Red bollworm *Pectinophora gossypiella* (Saunders); 5. Wheat moth *Sitotroga cerealella* (olivier); 6. American serpentine leafminer *Liriomyza sativae* (Blanchard); 7. Three-leaf serpentine leafminer *Liriomyza trifolii* (Burgess); 8. South American serpentine leafminer *Liriomyza huidobrensis* (Blanchard); 9. Onion leafminer *Liriomyza chinensis* (Kato); 10. Pea leafminer *Phytomyza horticola* (Gourean); 11. Diamondback moth *Plutella* 12. *Spodoptera xylostella* (L.); 13. *Spodoptera frugiperda* (JESmith); 14. *Liriomyza bryoniae* (Kaltenbach); 15. *Spodoptera exigua* (Hübner); negative control (ultrapure water); 16. *Spodoptera erinacea* (Fabricius); 17. *Helicoverpa armigera* (Hübner); 18. *Anatatrifolii* (Hufnagel); 19. *Mythimna separata* (Walker); 20. *Argyrogramma agnata* (Staudinger); 21. *Conogethes punctiferalis* (Guenée); 22. *Ostriniafurnacalis* (Guenée); 23. *Plodia lataniae* (Indian meal borer). 23. *Interpunctella interpunctella* (Hübener); 24. *Grapholita molesta* (Busck); 25. *Cydiapomonella* (L.); 26. *Grapholithafunebrana* (Treitschke); 27. *Hyphantria cunea* (Drury); Negative control (ultrapure water).

[0034] Figure 3Alignment results of different lock nucleic acid modification sites of Tuta-P in the tomato leafminer with the target binding sequences of the tomato leafminer (Tuta absoluta) and its closely related species, as well as leaf-eating / leaf-mining / fruit-boring pests such as the potato tuber moth (Phthorimaea operculella), the Guatemalan potato moth (Tecia solanivora), the tobacco stem borer (Scrobipalpa costella), and the beet armyworm (Spodopteralitura).

[0035] Figure 4 A schematic diagram of the structure and detection results of the single-target disposable nucleic acid test strip (JY0201).

[0036] Figure 5 The effect of primer sets A, B, C, and D for species-specific detection of tomato leafminer moth, and the dilution factor of RT-RAAnfo amplification products on the LFS detection results of the genomes of tomato leafminer moth and potato tuber moth.

[0037] Figure 6 The effect of primer addition volume in the RT-RAA-LFS detection results of the crude nucleic acid lysate of first-instar larval samples of *Tomato leafminer* on the species-specific detection primer set B in the RT-RAA-LFS amplification system.

[0038] Figure 7 The effect of the MgAc2 addition volume in the RT-RAA-LFS detection results of the crude nucleic acid lysate of first instar larval samples of *Tomato leafminer* on the species-specific detection primer set B.

[0039] Figure 8 The effect of the volume of crude nucleic acid lysis buffer added to the RT-RAA-LFS detection results of crude nucleic acid lysis buffer in the RT-RAA-LFS amplification system of primer group B for species-specific detection of tomato leafminer moth.

[0040] Figure 9The optimal RTRAA-LFS detection system for species-specific tomato leafminer was used to detect tomato leafminer and its sympatric or closely related species using RT-RAA-LFS. The detected species were: 1. *Tuta absoluta* (Meyrick); 2. *Phthorimaea operculella* (Zeller); 3. *Brachmia macroscopa* (Meyrick); 4. *Pectinophora gossypiella* (Saunders); 5. *Sitotroga cerealella* (olivier); 6. *Liriomyza sativae* (Blanchard); 7. *Liriomyza trifolii* (Burgess); 8. *Liriomyza huidobrensis* (Blanchard); 9. *Liriomyza chinensis* (Kato); 10. *Phytomyza* (Pea leafminer). 11. *Plutellaxylostella* (L.) horticola; 12. *Spodoptera frugiperda* (JESmith); 13. *Liriomyza bryoniae* (Kaltenbach); 14. *Spodoptera exigua* (Hübner); 15. *Spodoptera litura* (Fabricius); 16. *Helicoverpa armigera* (Hübner); 17. *Anata trifolii* (Hufnagel); 18. *Mythimna separata* (Walker); 19. *Argyrogramma* (Silver-striped Noctuid Moth) 20. *Agnata* (Staudinger); 21. *Conogethespunctiferalis* (Guenée); 22. *Ostriniafurnacalis* (Guenée); 23. *Plodia interpunctella* (Hübener); 24. *Grapholita molesta* (Busck); 25. *Cydiapomonella* (L.); 26. *Grapholithafunebrana* (Treitschke); 27. *Hyphantria cunea* (Drury); Negative control (1× nucleic acid lysis buffer).

[0041] Figure 10 Sensitivity analysis of the detection of adult tomato leafminer genomic solution and its gradient dilutions using primer set B for species-specific detection of tomato leafminer.

[0042] Figure 11 Sensitivity analysis of the detection of tomato leafminer target gene plasmids and their gradient dilutions RT-RAA-LFS using primer set B for species-specific detection of tomato leafminer.

[0043] Figure 12 Sensitivity analysis of RT-RAA-LFS detection of crude nucleic acid lysates and their gradient dilutions of Tomato Leafminer using primer set B for species-specific detection of Tomato Leafminer. Among them, I represents eggs, II represents first instar larvae, III represents second instar larvae, IV represents third instar larvae, V represents fourth instar larvae, VI represents pupae, and VII represents adults.

[0044] Figure 1. RT-RAA-LFS detection results of 1350 randomly collected insect samples from the field. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0046] The insect samples and their corresponding DNA involved in this invention are preserved in the National Key Laboratory of Integrated Pest Management, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Tomato leaf samples infested with the tomato leafminer were collected in Beijing, Gansu, Xinjiang, Yunnan, Hainan, Shanxi, Jiangxi, and Hubei provinces from 2022 to 2023. Leaves with irregular translucent spots, wrinkled and dried leaves, or fruits with holes or deformities were selected. After collection, the samples were flash-frozen in liquid nitrogen and stored at -70°C, or immersed in 99.7% anhydrous ethanol.In the RAA-specific primer screening experiment for the tomato leafminer, the following species were selected: *Tuta absoluta* (Meyrick), *Phthorimaea operculella* (Zeller), *Brachmia macroscopa* (Meyrick), *Pectinophora gossypiella* (Saunders), *Sitotroga cerealella* (Olivier), *Liriomyza sativae* (Blanchard), *Liriomyza trifolii* (Burgess), *Liriomyza huidobrensis* (Blanchard), *Liriomyza chinensis* (Kato), *Phytomyza horticola* (Gourean), *Plutella xylostella* (L.), *Spodoptera frugiperda* (JESmith), and *Liriomyza*. *Spodoptera bryoniae* (Kaltenbach), *Spodoptera exigua* (Hübner), *Spodoptera litura* (Fabricius), *Helicoverpa armigera* (Hübner), *Anata trifolii* (Hufnagel), *Mythimna separata* (Walker), *Argyrogrammaagnata* (Staudinger), *Conogethespunctiferalis* (Guenée), *Ostriniafurnacalis* (Guenee), *Plodia interpunctella* (Hübener), *Grapholita molesta* (Busck), *Cydia pomonella* (L.), *Grapholithafunebrana* (Treitschke), and *Hyphantria* (American white moth). The cunea (Drury) was used as the experimental subject. DNA was extracted from the worm tissue as a template for amplification, and an equal volume of ultrapure water was used as a negative control.

[0047] Using the rapid universal genomic DNA extraction kit (3105050) from Hangzhou Xinjing Biological Reagent Development Co., Ltd., and following the kit instructions, genomic DNA was extracted from adult tomato leafminer moths and their closely related species, as well as leaf-eating / leaf-mining / fruit-boring pests such as potato tuber moth, sweet potato leafminer, red bollworm, wheat moth, American serpentine leafminer, trifoliate leafminer, South American serpentine leafminer, onion leafminer, pea leafminer, diamondback moth, fall armyworm, beet armyworm, cotton bollworm, spiny armyworm, armyworm, silver-striped armyworm, peach fruit borer, Asian corn borer, Indian meal borer, pear fruit moth, and adult codling moth. The nucleic acid lateral flow chromatography test strips are from Beijing Baoying Tonghui Biotechnology Co., Ltd.

[0048] To evaluate whether the RAA-LFS system can be used as a rapid and specific detection method for tomato leafminer eggs, pupae, larvae, and adults, the genomic DNA solution of the above samples was used as an amplification template, with an equal volume of ultrapure water as a negative control. Alternatively, a single egg or a first-instar larva (approximately 0.5 cm × 0.5 cm in leaf area) can be placed on the cap of a 1.5 mL Eppendorf tube. The tissue is thoroughly ground using a 1 mL pipette tip with a rounded tip. 50 μL of Starsparkle lysis buffer is added to the ground tissue, and after gentle aspiration several times, 2 μL of the nucleic acid lysis supernatant is used as a template for RT RAA amplification. Using a pipette tip or tweezers, a leaf containing a relatively large larva, pupa, or adult is carefully placed into a 1.5 mL Eppendorf tube. The tissue is thoroughly ground using a 1 mL pipette tip with a rounded tip, 200 μL of Starsparkle lysis buffer is added, and after gentle aspiration several times, 2 μL of the nucleic acid lysis supernatant is used as a template for RT RAA amplification. The sample lysis buffer can be tested immediately or stored at -70°C for more than 3 months.

[0049] Example 1: Primer design and screening for species-specific detection of tomato leafminer

[0050] After extensive sequence analysis, our team selected the tomato leafminer cytochrome c oxidase subunit I gene (COX1), tRNA-Leu, and cytosol-synthesized subunit II gene (COX2) as target genes. Following analysis of the target gene sequence characteristics, we designed species-specific amplification primers using Primer Premier 5.0 software. The primer design followed these principles.

[0051] ① The amplification primers are about 20-30 nt in length, and the annealing temperatures of different primers should be as consistent or similar as possible; ② The amplification product is about 200-500 bp in length, and similar sequences are only found in the mitochondrial genomes of the tomato leafminer and the potato tuber moth.

[0052] ② The primer amplification region sequences were compared using the BLAST function of the National Center for Biotechnology Information (NCBI) to preliminarily identify the primer amplification specificity. The identified primers were synthesized by General Biosystems (Anhui) Co., Ltd., with a purity level of HPLC. The specific primer sequences are shown in Table 1.

[0053] Table 1. Primers for amplification using the RAA basic primer set for the species-specific primers of the tomato leafminer.

[0054]

[0055] use RAA Nucleic Acid Amplification Kit (Basic Version JY0203) uses genomic DNA solutions from the tomato leafminer and potato tuber moth as templates for RAA amplification. RAA amplification is performed using both forward and reverse primers of primer pairs 1-4. The amplification system (single sample / reaction) is as follows:

[0056] Element Volume (μL) Plant sample lysate or total RNA solution 2 Basic buffer 29.4 Forward primer (10 μM) 2.1 Reverse primer (10 μM) 2.1 <![CDATA[ddH2O]]> The total system value is 47.5.

[0057] After the reaction solution is prepared, add 2.5 μL of 280 mM MgAc2 to each 0.2 mL Eppendorf tube and mix thoroughly; place the 0.2 mL Eppendorf tube on a PCR instrument with a heated lid and incubate at 39 °C for 30 min.

[0058] After the RAA reaction is complete, remove the reaction tubes. Add 100 μL of phenol / chloroform (1:1) to each reaction tube, vortex thoroughly, and centrifuge at 12000 rpm for 10 min (this step can be done by vigorous vortexing). Mix 10 μL of the supernatant with 2 μL of 6× Loading Buffer, load the mixture onto a 1.5% agarose gel, and electrophoresis at 200 V for 15 min. Stop electrophoresis when bromophenol blue has moved to 2 / 3 of the gel. Stain with EB for 5 min, observe and photograph under UV light.

[0059] like Figure 1As shown, with the addition of 2 μL of genomic DNA solutions from *Tomato leafminer* and *Potato tuber moth*, RAA amplification was performed in the *Tomato leafminer* genomic DNA solutions using primer pairs 1 (Tuta-F and Tuta-R1), 2 (Tuta-F and Tuta-R2), 3 (Tuta-F and Tuta-R), and 4 (Tuta-F and Tuta-R4), respectively. Primers 1 and 4 showed no amplification bands in *Potato tuber moth*, while primer pair 2 showed a weaker amplification band than primer pair 3. Primer pair 3 formed amplification products in both *Tomato leafminer* and *Potato tuber moth* genomic DNA solutions, and the electrophoretic bands of the amplified products were clear, bright, and free of impurities. The band size was consistent with expectations, making it suitable as a dedicated primer for detection (see [link to relevant documentation]). Figure 1 The amplification products of primer set 3 in the genomic DNA solutions of tomato leafminer and potato tuber moth were cloned, sequenced, and aligned to reveal the target sequence.

[0060] RAA amplification of genomic DNA solutions from the tomato leafminer and its closely related species, as well as leaf-eating / leaf-mining / fruit-boring pests, using the tomato leafminer-specific primers Tuta-F / Tuta-R, showed that only the tomato leafminer and the potato tuber moth produced specific amplification bands. However, no amplification bands were observed for the sweet potato leafminer, red bollworm, wheat moth, American serpentine leafminer, trifoliate leafminer, South American serpentine leafminer, onion leafminer, pea leafminer, diamondback moth, tomato leafminer, beet armyworm, negative control, cotton bollworm, spiny armyworm, armyworm, silver-striped armyworm, peach fruit borer, Asian corn borer, Indian meal borer, pear fruit moth, codling moth, plum fruit moth, and fall webworm (see [link to relevant documentation]). Figure 2 ).

[0061] Experiment Example 2: Preparation and Composition of Sample Comparison Disc for Species-Specific Detection of Tomato Leafminer

[0062] The amplification products of primer pair 3 (Tuta-F and Tuta-R) for the tomato leafminer were transformed into plasmids with correct cloning and sequencing, and then subjected to overnight culture for plasmid extraction. The OD of the recombinant plasmids was measured using a UV spectrophotometer. 260 OD 280 and OD 260 / OD 280 The values ​​were measured and repeated three times to determine the plasmid DNA concentration and purity.

[0063] The copy number of the plasmid is obtained using the following formula:

[0064] Copy number = plasmid concentration × 6.02 × 10 23 / (660 × total plasmid length)

[0065] Calculate the copy number and dilute to 5.1 × 10⁻⁶. 8Copy / μL, store at –20℃ for later use. Dilute the pre-determined recombinant plasmid to 5.1 × 10⁻⁶. 6 Copy / μL, followed by 10-fold serial dilutions, yielded 5.1 × 10⁻⁶ μL. 6 Copy / μL, 5.1×10 5 Copy / μL, 5.1×10 4 Copy / μL, 5.1×10 3 Copy / μL, 5.1×10 2 Copy / μL, 5.1×10 1 copies / μL and 5.1×10 0 A copy / μL plasmid dilution was used as a template for subsequent amplification.

[0066] Experiment Example 3: RAA-LFS Detection of Positive Plasmids for Species-Specific Detection of Tomato Leafminer

[0067] For the amplification region of the tomato leafminer-specific primers Tuta-F / Tuta-R in Example 1, species-specific probe sequences were designed using PrimerPremier 5.0 software. The probe sequences were located in the middle segment of the amplification primers, and different numbers (0, 3, 5, and 7) of tetrahydrofuran were used to modify the tomato leafminer species-specific haplotype sites. The primer design followed the principles of: ① probe length approximately 30–45 nt; ② probe sequence species specificity.

[0068] The primer amplification region sequences were compared using the BLAST function of the National Center for Biotechnology Information (NCBI) to preliminarily identify the primer amplification specificity. The identified primers were synthesized by General Biosystems (Anhui) Co., Ltd., with a purity level of HPLC. The specific primer sequences, probes, and amplification reverse primer labeling are shown in Table 2 below.

[0069] The probes used in the experiment have the following characteristics: ① Designed in the forward direction, i.e., in the same direction as the forward primer; ② Length between 46 and 52 nt; ③ Phosphorylation modification at the 3′ end to inhibit DNA chain elongation; ④ Added cleavage sites within the sequence, which are cleaved by nfo enzyme when the probe binds to the template DNA, initiating DNA chain elongation; ⑤ Added locking nucleotide modification to recognize single nucleotide mutations; ⑥ Biotin modification at the 5′ end, so that the double-stranded amplification product of the probe and the reverse (FITC-modified) primer can be detected by nucleic acid test strips.

[0070] Table 2. Primer sets for RT RAA-nfo amplification of tomato leafminer and potato tuber moth.

[0071]

[0072] Using genomic DNA from the tomato leafminer and potato tuber moth as templates, The RAA nucleic acid amplification kit (test strip method) (JY0204) is used to prepare the RAA reaction system for primer sets A to D (single sample / reaction). The system is as follows:

[0073] Components Dosage (μL) Basic buffer 29.4 Forward primer (10 μM) 2.1 Probe primers (10 μM) 0.6 Reverse primer (10 μM) 3.0 Recombinant plasmid solution 2 Add water to make up the volume. 47.5

[0074] The sample addition order is as follows: negative control sample (genomic DNA solution replaced with an equal volume of ultrapure water), 2 μL genomic DNA solution. After each sample addition, the tube cap must be immediately closed to prevent aerosol contamination. Mix the above reaction system thoroughly and add it to the basic reaction unit. Ensure the lyophilized powder is fully dissolved; note that this step should not be done by vigorous vortexing. Open the reaction unit and add 2.5 μL of 280 mM MgAc2 to each 0.2 mL Eppendorf tube. Mix thoroughly and collect by centrifugation. Note that this step should not be done by vigorous vortexing.

[0075] The reaction tube was placed at 39°C for 16 min. After the RTRAA reaction was complete, the Eppendorf tube was opened, the amplified product was aspirated into a new Eppendorf tube, labeled, and diluted 25, 50, and 100 times before LFS detection.

[0076] A schematic diagram of the structure of the single-target disposable nucleic acid test strip (JY0201) is shown below. Figure 4 As shown, insert the test strip into the immersion area (marked with a blue arrow pointing upwards) of the Eppendorf tube, ensuring the liquid level does not exceed the MAX indicator line of the immersion area. Wait until the entire interpretation area is saturated (approximately 30-60 seconds), then lay the test strip flat for 1 minute, waiting for the red band to appear. Read the test result directly based on the color development of the test strip. Observe the results within 10 minutes; results after 10 minutes are invalid.

[0077] like Figure 5 As shown, primer sets A (Tuta-F+Tuta-P1+Tuta-R) and B were used for the tomato leafminer moth.

[0078] After RTRAAnfo amplification of (Tuta-F+Tuta-P+Tuta-R), C(Tuta-F+Tuta-P3+Tuta-R), and D(Tuta-F+Tuta-P4+Tuta-R), the conditions for meeting the negative control and the negative LFS detection of the potato tuber moth genome and the positive LFS detection of the tomato leafminer genome are as follows: LFS detection of the 100-fold dilution of the RTRAA amplification product of primer group B, LFS detection of the 25-fold and 50-fold dilutions of the RTRAA amplification product of primer group C, and LFS detection of the 25-fold dilution of the RRAA amplification product of primer group D.

[0079] Comparing the colorimetric results of different primer sets at different dilutions, it was found that the LFS detection band of the 100-fold dilution of primer set B RT RAA amplification product was significantly darker than the LFS bands of the 25-fold and 50-fold dilutions of primer set C RTRAA amplification product, and the LFS band of the 25-fold dilution of primer set D RTRAA amplification product. Considering the primer modification cost, the 100-fold dilution of primer B RT RAAnfo amplification was selected for LFS detection, and this primer was used for subsequent RTRAA-LFS detection and optimization of the RTRAA nucleic acid amplification system.

[0080] Example 4: Optimization of the RAAnfo-LFS detection system using species-specific detection primer set B for tomato leafminer.

[0081] The test strip's detection product is a double-stranded amplification product of the probe after being digested with nfo enzyme and the reverse primer (modified with FITC). Therefore, theoretically, increasing the amount of reverse primer added can help improve the sensitivity of the RAA nucleic acid test strip. However, adding too much reverse primer can also increase the probability of reverse primer and probe primer dimer formation, leading to false positive results.

[0082] The crude form of nucleic acid lysis buffer and its gradient dilutions from first-instar larvae of the tomato leafminer were used as templates for RTRAA amplification. The RT-RAA nucleic acid amplification kit (test strip method) (JY0204) was used to prepare the RAA reaction system (single sample / reaction). The ratio of forward:probe:reverse primer addition was 7:2:10. The primer mixture was prepared according to this ratio, and the addition volume of different primer sets within the RT-RAA amplification system was adjusted as shown in Table 3. For each system, 2 μL of the crude nucleic acid lysis buffer or its gradient dilution was used as a template, and negative control samples were set for testing. After adding samples, the tube caps should be immediately closed to avoid aerosol contamination. The above reaction system was mixed thoroughly and added to the basic reaction unit. The lyophilized powder was fully dissolved; note that vigorous vortexing should not be used for mixing in this step. The reaction unit was opened, and 2.5 μL of 280 mM MgAc2 was added to each 0.2 mL Eppendorf tube. The mixture was thoroughly mixed and collected by centrifugation. Note that vigorous vortexing should not be used for mixing in this step.

[0083] Table 3. Adjustment of reverse primer addition amount in the RT-RAA amplification system for species-specific detection of tomato leafminer (Primer set B)

[0084]

[0085]

[0086] Open the reaction unit and add 2.5 μL of 280 mM MgAc2 to each 0.2 mL Eppendorf tube. Mix thoroughly and collect by centrifugation. Note that this step should not be done by vigorous vortexing. Incubate the reaction tubes at 39 °C for 16 min. After the RAA reaction is complete, open the Eppendorf tubes, aspirate the amplification product into a new Eppendorf tube, label it, and dilute it 100-fold. Perform a test strip test to determine the optimal addition volume for primer set B RT-RAA amplification.

[0087] like Figure 6 As shown, when using the crude lysate and its gradient dilutions of nucleic acid lysate from first-instar larvae of the tomato leafminer as templates for RT RAA amplification and performing RT RAA-LFS detection, with a primer set B addition volume of 5.7 μL, the crude lysate, 10-fold gradient dilution, 100-fold gradient dilution, and 1000-fold gradient dilution of the nucleic acid lysate from first-instar larvae of the tomato leafminer all tested positive, even when the negative control and potato tuber moth samples were negative. Therefore, the addition volume of primer set B for the tomato leafminer was determined to be 5.7 μL.

[0088] Table 4. Adjustment of primer set B for species-specific detection of tomato leafminer and the amount of MgAc2 added in RT-RAA amplification.

[0089]

[0090] Prepare the reaction solution according to the volumes indicated in Table 4, and add the corresponding volume of 280 mM MgAc2. Mix thoroughly and collect by centrifugation. Note that this step should not involve vigorous vortexing. Place the reaction tube at 39°C for 16 min. After the RT-RAA reaction is complete, open the Eppendorf tube, aspirate the amplification product into a new Eppendorf tube, label it, dilute it 100-fold, and perform a test strip test to determine the optimal volume of MgAc2 added for primer set B RT-RAA amplification.

[0091] like Figure 7As shown, when using the crude lysate of nucleic acid from first-instar larvae of the tomato leafminer and its gradient dilutions as templates for RT RAA amplification and performing RT RAA-LFS detection, with primer set B added at a volume of 5.7 μL and MgAc2 added at a volume of 2.5 μL, the crude lysate of nucleic acid from first-instar larvae of the tomato leafminer, its 10-fold gradient dilution, its 100-fold gradient dilution, and its 1000-fold gradient dilution were all detected as positive, even when the negative control and potato tuber moth were negative. Therefore, the optimal addition volume of primer set BMgAc2 for the tomato leafminer was determined to be 2.5 μL.

[0092] Table 5. Adjustment of the amount of crude extract of RT-RAA amplified nucleic acid lysate from primer set B for species-specific detection of tomato leafminer.

[0093]

[0094] Prepare the reaction solution according to the volumes indicated in Table 5, and add the corresponding volume of 280 mM MgAc2. Mix thoroughly and collect by centrifugation. Note that this step should not involve vigorous vortexing. Place the reaction tube at 39°C for 16 min. After the RT-RAA reaction is complete, open the Eppendorf tube, aspirate the amplification product into a new Eppendorf tube, label it, and dilute it 100-fold. Perform a test strip test to determine the optimal volume of crude nucleic acid lysate extract for primer set B RT-RAA amplification.

[0095] like Figure 8 As shown, when using the crude nucleic acid lysis buffer and its gradient dilutions from first-instar larvae of the tomato leafminer as templates for RT-RAA amplification and performing RT-RAA-LFS detection, with primer set B added at a volume of 5.7 μL, MgAc2 added at a volume of 2.5 μL, and crude nucleic acid lysis buffer added at a volume of 2 μL, the crude nucleic acid lysis buffer, 10-fold gradient dilution, 100-fold gradient dilution, and 1000-fold gradient dilution of the crude nucleic acid lysis buffer from first-instar larvae of the tomato leafminer all tested positive, even when the negative control and potato tuber moth samples were negative. Therefore, the optimal addition volume of the crude nucleic acid lysis buffer from primer set B of the tomato leafminer was determined to be 2 μL.

[0096] Example 5: Specificity analysis of the primer set BRTA-LFS for the species-specific detection of tomato leafminer.

[0097] After determining the optimal addition levels of each component in the tomato leafminer species-specific detection system, the system was used to perform RT-RAA-LFS detection on the tomato leafminer and its sympatric or closely related species. Figure 9The test results show that the detection system can accurately distinguish the tomato leafminer and its 25 sympatric or closely related species.

[0098] Example 6: Sensitivity analysis of the BRT RAA-LFS primer set for species-specific detection of tomato leafminer.

[0099] Using the tomato leafminer adult genome solution as a template, and the plasmid corresponding to the amplified fragment of the tomato leafminer species-specific primer set B (Tuta-F+Tuta-P+Tuta-R) as a template, the optimal RT RAAnfo amplification system determined in Experiment 4 was used to complete RAAnfo amplification of 1.6 ng, 160 pg, 16 pg, 1.6 pg, 160 fg, and 16 fg genome solutions. The amplified products were diluted 100-fold and then subjected to LFS detection. The detection threshold of the tomato leafminer primer set B RT RAAnfo-LFS was determined to be 1.6 pg (… Figure 10 ).

[0100] Using the plasmid corresponding to the amplified fragment of the tomato leafminer species-specific primer set B (Tuta-F+Tuta-P+Tuta-R) as a template, and the optimal RT RAAnfo amplification system determined in Experiment 4, 5.1 × 10⁻⁶ amplifications were completed. 6 Copy / μL, 5.1×10 5 Copy / μL, 5.1×10 4 Copy / μL, 5.1×10 3 Copy / μL, 5.1×10 2 Copy / μL, 5.1×10 1 copies / μL and 5.1×10 0 RAAnfo amplification was performed using a serial dilution buffer of the corresponding plasmid at a concentration of 1 copy / μL. The amplified product was then diluted 100-fold and subjected to LFS detection. The detection threshold for the tomato leafminer primer group B RT-RAAnfo-LFS was determined to be 51 copies. Figure 11 ).

[0101] Example 7: Detection of Tomato Leafminer Samples at Different Instars using Primer Set B RT RAA-LFS for Species-Specific Detection of Tomato Leafminer

[0102] Using crude lysates and gradient dilutions of nucleic acid lysates from different instars of the tomato leafminer as templates, and employing the optimal RT-RAAnfo amplification system determined in Experiment 4, RT-RAA nfo amplification of eggs, first-instar larvae, second-instar larvae, third-instar larvae, fourth-instar larvae, pupae, and adults was completed. The amplified products were then diluted 100-fold and subjected to LFS detection to determine the detection threshold of the tomato leafminer primer set BRT-RAAnfo-LFS for samples of different instars. Figure 12 ).

[0103] The single oocyte was processed as follows: a single oocyte was placed on the cap of a 1.5 mL Eppendorf tube and thoroughly ground using a 1 mL pipette tip with a rounded tip. 50 μL of Starsparkle lysis solution was added to the thoroughly ground tissue, and the tissue was gently aspirated and pipetted several times. Then, 2 μL of the lysate supernatant was transferred to the RT RAA amplification system.

[0104] The treatment of first-instar larvae involved carefully placing an infected leaf (approximately 0.5cm x 0.5cm square) containing the larvae onto the cap of a 1.5mL Eppendorf tube using a punch or tweezers. The leaf was then thoroughly ground using a 1mL pipette tip with a rounded tip. 50μL of Starsparkle lysis solution was added to the ground tissue, and the mixture was gently aspirated several times. Finally, 2μL of the lysate supernatant was transferred to the RT RAA amplification system.

[0105] The treatment of second-instar larvae, third-instar larvae, fourth-instar larvae, and pupae was as follows: Using a pipette tip or forceps, carefully place the leaf containing the larvae or pupae into a 1.5 mL Eppendorf tube, and thoroughly grind it using a 1 mL pipette tip with a rounded tip. Add 200 μL of Starsparkle lysis solution to the thoroughly ground tissue, gently aspirate and pipette several times, and then aspirate 2 μL of the lysate supernatant into the RT RAA amplification system.

[0106] The adult worms were carefully placed into a 1.5 mL Eppendorf tube using forceps, and thoroughly ground using a 1 mL pipette tip with a rounded tip. 200 μL of Starsparkle lysis solution was added to the thoroughly ground tissue, and the tissue was gently aspirated several times. Then, 2 μL of the lysate supernatant was transferred to the RT RAA amplification system.

[0107] After processing the samples of different ages, the crude nucleic acid extracts of different samples were serially diluted using crude lysis buffer. It was found that single eggs and pupae could be detected down to a 100-fold dilution of the crude nucleic acid extract, while first-instar larvae, second-instar larvae, third-instar larvae, fourth-instar larvae and adults could be detected down to a 1000-fold dilution of the crude nucleic acid extract, indicating that the established detection system has excellent detection sensitivity.

[0108] Example 8: Detection of field samples using the tomato leafminer species-specific primer set B RT RAA-LFS.

[0109] Using the crude form of nucleic acid lysis buffer from randomly sampled insects in the field as a template, the optimal RTRAA nfo amplification system determined in Experiment 4 was used to complete the RTRAA nfo amplification of the samples. After nucleic acid extraction, a volume equivalent to 2 μL of nucleic acid lysis buffer was used for qPCR reaction. The primers used were those from Wang et al. (Y.Wang, X.Tian, ​​H.Wang, C.). J. Arnó, S. Wu, et al., Genetic diversity and genetic differentiation pattern of Tuta absoluta across China, Entomologia Generalis, (2023)., to verify the accuracy of RT-RAA test results.

[0110] Results of RT RAA-LFS analysis of 50 field samples are as follows Figure 13 As shown in Table 6, the RT-RAA-LFS detection results were completely consistent with the qPCR results of the nucleic acid extract of the insect sample nucleic acid lysate. Furthermore, the intensity of the RT-RAA-LFS color bands was related to the qPCR Cq value of the nucleic acid extract of the insect sample nucleic acid lysate, indicating that this system can achieve rapid field identification of whether insect samples of different ages are tomato leafminer moths by means of rapid sample lysis.

[0111] Table 6. RT-RAA-LFS and qPCR detection of 650 field-collected insect samples

[0112]

[0113]

[0114] In summary, this application has screened a primer set that can be used for field detection of tomato leafminer through a series of verifications. This primer set can effectively amplify the mitochondrial target gene of tomato leafminer, with high specificity and sensitivity, and no cross-reaction with other sympatric insects or closely related species. It can be used for rapid field identification of tomato leafminer, which is of great significance for the effective prevention and control of this major invasive species and a Class I crop pest in my country.

[0115] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A type of tomato leafminer Tuta absoluta mitochondria COI A barcode species-specific RT-RAA detection primer set, characterized in that, The primer set includes the tomato leafminer. Tuta absoluta Species-specific detection primers and detection probe Tuta-P; among which, The tomato leafminer Tuta absoluta The detection primers include the forward primer Tuta-F and the reverse primer Tuta-R; The sequence of the forward primer Tuta-F is: 5´-TATTTATATTAATTATTATTTGAGAA-3´. The sequence of the reverse primer Tuta-R is: 5´-GGAGAAGTCCCATTTTGAAGATT-3´; The detection probe Tuta-P has the sequence 5´-AATGATATCAATTA. T TACC C CC C The reverse primer Tuta-R is modified with FITC at its 5' end, and the detection probe Tuta-P is modified with biotin at its 5' end. Tuta-P has phosphorylation modification at its 3' end, H is tetrahydrofuran, and all underlined bases in the Tuta-P sequence are modified with locked nucleotides.

2. A method for detecting tomato leafminer Tuta absoluta The reagent is characterized by, The reagent contains the primer set as described in claim 1.

3. A method for detecting tomato leafminer Tuta absoluta The reagent kit is characterized by, The kit includes the primer set as described in claim 1.

4. The reagent according to claim 2 or the kit according to claim 3 for detecting tomato leafminer in biological samples. Tuta absoluta In applications where tomato leafminer can be detected... Tuta absoluta If the mitochondrial COI barcode sequence is found, then the biological sample is infected with the tomato leafminer moth.

5. A method for detecting tomato leafminer based on RT-RAA-lateral flow chromatography. Tuta absoluta The method is characterized by, Includes the following steps: 1) Perform lysis on the sample or extract genomic DNA from the biological sample to be tested; 2) Using the sample lysis buffer or extracted genomic DNA obtained in step 1) as a template, perform RT-RAA amplification using the primer set described in claim 1; 3) Use lateral flow chromatography test strips to detect amplification products, observe the color development of the bands on the lateral flow chromatography test strips, and determine whether the sample contains tomato leafminer.

6. The method according to claim 5, characterized in that, The color development of the bands on the side-flow chromatography test strip is observed to determine whether the sample contains tomato leafminer. Tuta absoluta The test results are as follows: If the test strip shows one band at the control line and no band at the test line, the result is negative, indicating that the sample does not contain or is not infected with tomato leafminer. If the test strip shows two bands, one at the control line and one at the test line, the result is positive, indicating that the sample contains or is infected with tomato leafminer. If the control line of the test strip does not show color, the test is invalid and the test strip needs to be replaced and the test repeated.

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