A primer pair for identifying wild-harvested ants and application thereof

Abstract

This invention discloses a primer pair for identifying the barbaric harvester ant and its application, relating to the field of molecular biology. The invention discloses primer pairs with nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:6. It overcomes the limitations of traditional morphological detection and can effectively distinguish the barbaric harvester ant from nine closely related species (*C. spp.*, *C. brevicornu*, *C. spp.*, *C. spp.*, *C. spp.*, *C. spp.*, *C. spp.*, *C. spp.*, *C. spp.*, *C. spp.*, *C. spp.*, *C. spp.*, and *C. spp.*). It is suitable for detecting different developmental stages of the barbaric harvester ant, achieving rapid, accurate, and stable detection with a sensitivity as low as 0.01 ng / μL.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a primer pair for identifying wild harvester ants and its application. Background Technology

[0002] The Messor ant, belonging to the family Formicidae and genus Messor in the order Hymenoptera, feeds primarily on plant seeds. They are adept at collecting herbaceous plant seeds, processing them into "seed bread," and storing it in their nests for consumption during the cold season. They can easily damage vegetation. Colonies can range in size from several thousand to tens of thousands of individuals, with a clear division of labor. They are fierce and skilled in collective combat. The queen has a long lifespan and high reproductive capacity; a mature colony can produce a large number of reproductive ants annually. They exhibit strong adaptability and reproductive capabilities.

[0003] Harvester ants are frequently detected during import and export inspections and quarantine. However, traditional morphological identification methods require a high level of expertise from the personnel involved, making it difficult to meet the needs of customs quarantine departments for accurate and rapid identification of harvester ants. Therefore, developing a method for the efficient, accurate, and sensitive identification of harvester ants is of paramount importance. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a primer pair for identifying Savage Harvester Ant, which has high specificity and high sensitivity, and can accurately identify Savage Harvester Ant.

[0005] The present invention also provides a reagent for identifying wild harvester ants.

[0006] The present invention also provides a kit for identifying wild harvester ants.

[0007] The present invention also provides applications of the above primer pairs, reagents or kits.

[0008] The present invention also provides a method for detecting or assisting in the detection of whether a sample to be tested is a Savage Harvester Ant or whether it contains Savage Harvester Ants.

[0009] The present invention also provides a system for detecting barbaric harvester ants.

[0010] According to a first aspect of the present invention, a primer pair for identifying wild harvester ants includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 6.

[0011] The primer pairs according to embodiments of the present invention have at least the following beneficial effects:

[0012] The primer pairs used in this embodiment overcome the limitations of traditional morphological detection, effectively distinguishing *Ictalus savageus* from nine closely related species (*Ictalus big-headed*, *Ictalus bournei*, *Ictalus blazei*, *Ictalus artisans*, *Ictalus orientalis*, *Ictalus stigmatus*, *Ictalus robustus*, *Ictalus industriousus*, and *Ictalus laurentii*). They are suitable for detecting different developmental stages of *Ictalus savageus*, achieving rapid, accurate, and stable detection with a sensitivity as low as 0.01 ng / μL. This not only lays the foundation for the molecular-level classification of the genus *Ictalus* in the family Formicidae of the order Hymenoptera and the rapid identification of closely related species, but also has extremely important significance for protecting agricultural and forestry production safety and maintaining ecological balance.

[0013] According to some embodiments of the present invention, at least one of the upstream primer and the downstream primer has a detectable marker.

[0014] According to some embodiments of the present invention, the detectable marker includes, but is not limited to, fluorescent markers.

[0015] According to some embodiments of the present invention, the fluorescent label includes at least one of FAM, ROX, CY5, HEX, JOE, CY3, NED, TAMRA, TAXAS RED, VIC, and TET.

[0016] A reagent for identifying barbaric harvester ants according to a second aspect of the present invention comprises the primer pair described in the first aspect embodiment.

[0017] A kit for identifying Savage Harvester Ants according to a third aspect of the present invention includes the primer pair described in the first aspect embodiment or the reagent described in the second aspect embodiment.

[0018] According to some embodiments of the present invention, the kit further includes a PCR amplification premix.

[0019] According to some embodiments of the present invention, the PCR amplification premix includes a stabilizer, DNA polymerase, and Mg. 2+ and at least one of dNTPs. The DNA polymerase includes, but is not limited to, Taq DNA polymerase.

[0020] The use of the primer pair described in the first aspect embodiment, the reagent described in the second aspect embodiment, or the kit described in the third aspect embodiment according to the fourth aspect embodiment of the present invention in any of A1)-A6):

[0021] A1) Distinguish between the Savage Harvester Ant and its closely related species;

[0022] A2) Prepare products that distinguish between Savage Harvester Ant and its closely related species;

[0023] A3) Identification or auxiliary identification of Savage Harvester Ants;

[0024] A4) Prepare products for identification or to assist in the identification of Savage Harvester Ants;

[0025] A5) To identify or assist in identifying whether the sample to be tested contains Savage Harvester Ants;

[0026] A6) Prepare products for identification or auxiliary identification of whether the sample to be tested contains Savage Harvester Ants;

[0027] The closely related species include at least one of the following: Bighead Harvester Ant, Bryce Harvester Ant, White-spined Harvester Ant, Craftsman Harvester Ant, Oriental Harvester Ant, Needle-haired Harvester Ant, Strong Harvester Ant, Industrious Harvester Ant, and Ebony Harvester Ant.

[0028] A method for detecting or assisting in the detection of whether a sample to be tested is a barbaric harvester ant or whether it contains barbaric harvester ants, according to a fifth aspect embodiment of the present invention, includes the following steps:

[0029] The genomic DNA of the sample to be tested is amplified by PCR using the primer pairs described in the first aspect embodiment, the reagents described in the second aspect embodiment, or the kit described in the third aspect embodiment to obtain amplification products; the amplification products are detected to assess whether the sample to be tested is a Savage Harvester Ant or whether it contains Savage Harvester Ants.

[0030] According to some embodiments of the present invention, the method is for non-disease diagnosis and treatment purposes.

[0031] According to some embodiments of the present invention, the annealing temperature for PCR amplification is 52℃-62℃. For example, it can be 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃ or 62℃.

[0032] According to some embodiments of the present invention, the reaction conditions for the PCR amplification are: denaturation at 94-96℃ for 30s-1 min; denaturation at 94-98℃ for 5-30s, annealing at 60-62℃ for 5-10s, extension at 68-72℃ for 10-30s, for 25-35 cycles; and total extension at 68-72℃ for 3-5 min.

[0033] According to some embodiments of the present invention, in the PCR amplification system, the concentration of the upstream primer is 0.1 μmol / mL to 0.4 μmol / mL; and the concentration of the downstream primer is 0.1 μmol / mL to 0.4 μmol / mL. For example, the concentrations of the upstream / downstream primers can be 0.1 μmol / mL, 0.15 μmol / mL, 0.2 μmol / mL, 0.25 μmol / mL, 0.3 μmol / mL, 0.35 μmol / mL, or 0.4 μmol / mL.

[0034] According to some embodiments of the present invention, the concentration of DNA polymerase in the PCR amplification system is 1.25 U / µL-1.5 U / µL. For example, the concentration of DNA polymerase can be 1.25 U / µL, 1.3 U / µL, 1.35 U / µL, 1.4 U / µL, 1.45 U / µL, or 1.5 U / µL.

[0035] According to some embodiments of the present invention, the Mg in the PCR amplification system 2+ The concentration is 1 mmol / mL-3 mmol / mL. For example: Mg 2+ The concentration can be 1 mmol / mL, 1.1 mmol / mL, 1.2 mmol / mL, 1.3 mmol / mL, 1.4 mmol / mL, 1.5 mmol / mL, 1.6 mmol / mL, 1.8 mmol / mL, 1.9 mmol / mL, 2 mmol / mL, 2.1 mmol / mL, 2.2 mmol / mL, 2.3 mmol / mL, 2.4 mmol / mL, 2.5 mmol / mL, 2.6 mmol / mL, 2.8 mmol / mL, 2.9 mmol / mL or 3 mmol / mL.

[0036] According to some embodiments of the present invention, the concentration of dNTPs in the PCR amplification system is 0.2 mmol / mL to 0.3 mmol / mL. For example, the concentration of dNTPs can be 0.2 mmol / mL, 0.21 mmol / mL, 0.22 mmol / mL, 0.23 mmol / mL, 0.24 mmol / mL, 0.25 mmol / mL, 0.26 mmol / mL, 0.27 mmol / mL, 0.28 mmol / mL, 0.29 mmol / mL, or 0.3 mmol / mL.

[0037] According to some embodiments of the present invention, the concentration of DNA in the test sample in the PCR amplification system is 10 ng / μL-100 ng / μL. For example, the concentration of DNA can be 10 ng / μL, 15 ng / μL, 20 ng / μL, 25 ng / μL, 30 ng / μL, 35 ng / μL, 40 ng / μL, 45 ng / μL, 50 ng / μL, 65 ng / μL, 70 ng / μL, 75 ng / μL, 80 ng / μL, 85 ng / μL, 90 ng / μL, 95 ng / μL, or 100 ng / μL.

[0038] According to some embodiments of the present invention, the criteria for evaluating whether the test sample is a Savage Harvester Ant or contains Savage Harvester Ant include: if the fragment size of the amplification product is 159-161 bp, then the test sample is a Savage Harvester Ant or contains Savage Harvester Ant; otherwise, the test sample is not a Savage Harvester Ant or does not contain Savage Harvester Ant.

[0039] A system for detecting barbaric harvester ants according to a sixth aspect embodiment of the present invention, the system comprising:

[0040] The amplification module is used to perform PCR amplification on the genomic DNA of the sample to be tested using the primer pairs described in the first aspect embodiment, the reagents described in the second aspect embodiment, or the kit described in the third aspect embodiment, to obtain amplification products;

[0041] The result evaluation module is used to detect the amplification product and evaluate whether the sample to be tested is a Savage Harvester Ant or whether it contains Savage Harvester Ants.

[0042] According to some embodiments of the present invention, the system further includes a DNA template extraction module for extracting genomic DNA from the sample to be tested.

[0043] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0044] Figure 1 The results show the specificity detection results for different primer pairs; A: Primer pair 1 (Mb-COIF334 and Mb-COIR547), B: Primer pair 4 (Mb-COIF341 and Mb-COIR576), C: Primer pair 3 (Mb-COIF121 and Mb-COIR280); Lane M: DL2000 Marker, Lane 1: Big-headed harvester ant MY003, Lane 2: Bryth's harvester ant MY004, Lane 3: Savage harvester ant MY006, Lane 4: Savage harvester ant MY013, Lane 5: White-thorn harvester ant MY007, Lane 6: Craftsman harvester ant MY008, Lane 7: Eastern harvester ant MY009, Lane 8: Big-headed harvester ant MY010, Lane 9: Needle-haired harvester ant MY011, Lane 10: Strong harvester ant MY014, Lane 11: Industrious harvester ant MY015, Lane 12: Ebony harvester ant MY016;

[0045] Figure 2 The results show the sensitivity detection results for primer pairs Mb-COIF121 and Mb-COIR280; Lane M: DL2000 Marker, Lane 1: 10 ng / μL, Lane 2: 1 ng / μL, Lane 3: 10-1 ng / μL, lane 4:10 -2 ng / μL, lane 5:10 - 3 ng / μL, lane 6:10 -4 ng / μL. Detailed Implementation

[0046] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0047] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0048] In the description of this invention, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0049] All samples used in this invention (as shown in Table 1) have been morphologically identified and reviewed by Lin Wei, a senior agronomist specializing in entomology.

[0050] Table 1

[0051]

[0052] Unless otherwise specified, the steps for preparing the DNA template in this embodiment of the invention are as follows:

[0053] Collector ant specimens were placed in 200 μL centrifuge tubes, and 60 μL of extraction buffer A (1% (w / w) SDS buffer, 50 mmol / L Tris·HCl, 25 mmol / L NaCl, 25 mmol / L EDTA) was added. The mixture was thoroughly ground with a smelting pipette tip, vortexed, and incubated at 65℃ for 45 min. After incubation, 60 μL of extraction buffer B (3 mol / L potassium acetate aqueous solution, pH 7.2) was added, and the mixture was slowly inverted and mixed. The mixture was then placed on ice for 1 h, centrifuged at 12000 rpm for 10 min, and the supernatant was transferred to a new 500 μL centrifuge tube. Two volumes of frozen anhydrous ethanol were added, and the mixture was mixed and placed at -20℃ for 1 h. The mixture was then centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and 400 μL of 75% (v / v) ethanol aqueous solution was added to wash the precipitate. The mixture was centrifuged at 12000 rpm for 10 min, the ethanol was dried, and the precipitate was dissolved in 50 μL of double-distilled water. After determining the DNA concentration and purity using a micro spectrophotometer, the samples were stored at -20℃ for later use.

[0054] Primer pair design

[0055] The COI sequences of *Anoptera savagei* and nine closely related species (*Anoptera savagei*, *Anoptera bryonyi*, *Anoptera blazei*, *Anoptera davidii*, *Anoptera orientalis*, *Anoptera stigmata*, *Anoptera robusta*, *Anoptera industriousa*, and *Anoptera cinnabarina*) were cloned and identified. Unexpectedly, the COI sequences were found to be highly conserved within the genus *Anoptera*, but also exhibited some degree of variation among species. These sequences can be used to study genetic differences among species of *Anoptera*, making them valuable marker genes for investigating phylogenetic relationships between different species of harvester ants. Therefore, four primer pairs (sequences shown in Table 2) were designed based on the COI gene fragment sequence of *Anoptera savagei* for identification.

[0056] Table 2

[0057]

[0058] Test case

[0059] 1. Specificity test.

[0060] DNA extracted from *Ageratium savageense*, *Ageratium big-headedense*, *Ageratium bournei*, *Ageratium blazei*, *Ageratium davidii*, *Ageratium davidii*, *Ageratium orientalis*, *Ageratium spicata*, *Ageratium styracifolium*, *Ageratium styracifolium*, *Ageratium styracifolium*, and *Ageratium styracifolium* was used as a DNA template. PCR reaction systems were prepared according to the system shown in Table 3, and PCR amplification was performed (PCR reaction conditions: 94℃ denaturation for 1 min; 98℃ denaturation for 10 s, 55-58℃ annealing for 10 s (annealing temperature set according to the Tm value of different primer pairs), 68℃ extension for 30 s, 35 cycles; 68℃ total extension for 5 min). 10 μL of PCR product and loading buffer were separated by 2% agarose TAE gel electrophoresis, stained with GeneRed nucleic acid dye, and observed, recorded, and photographed on a gel imaging system. The specificity of the four primer pairs for *Ageratium savageense* was evaluated by agarose gel electrophoresis. To ensure the reliability of the results, each experiment was repeated at least three times.

[0061] Table 3

[0062]

[0063] The results are as follows Figure 1 As shown.

[0064] The third primer pair amplified specific fragments in both samples of *Savage Harvester ant*, while no specific bands were amplified in the other nine closely related species. This indicates that primer pairs Mb-COIF121 and Mb-COIR280 have good specificity for *Savage Harvester ant*. The second primer pair, however, did not amplify any bands, and primer pairs 1 and 4 exhibited non-specific amplification and weak amplification bands.

[0065] Furthermore, the sequencing results of the PCR amplification products of primer pairs Mb-COIF121 and Mb-COIR280 completely matched the DNA sequence of the Savage Harvester Ant. This further demonstrates that primer pairs Mb-COIF121 and Mb-COIR280 can specifically amplify the Savage Harvester Ant.

[0066] 2. Annealing temperature test.

[0067] DNA extracted from wild harvester ants was used as a template. The PCR reaction system was prepared according to the system shown in Table 3, and PCR amplification was performed (PCR reaction conditions: 94℃ denaturation for 1 min; 98℃ denaturation for 10 s, 52–62℃ annealing for 10 s, 68℃ extension for 30 s, 35 cycles; 68℃ total extension for 5 min). 10 μL of PCR product and loading buffer were separated by 2% agarose TAE gel electrophoresis, stained with GeneRed nucleic acid dye, and observed, recorded, and photographed on a gel imaging system. Gradient PCR was used, with 8 temperature gradients per group. To ensure the reliability of the results, each experiment was repeated at least three times.

[0068] The third pair of primers showed good specificity in amplifying the target band of the Savage Harvester Ant at 52–62℃, and the PCR product obtained at 62℃ had clear bands and strong specificity.

[0069] 3. Sensitivity test.

[0070] DNA extracted from the wild harvester ant sample MY013 was used as a DNA template, and PCR reaction was performed according to the system described in Example 1 (final DNA template concentrations were 10 ng / μL, 1 ng / μL, 0.1 ng / μL, and 1×10⁻⁶ ng / μL). -2 ng / μL, 1×10 -3 ng / μL, 1×10 -4 The detection sensitivity of primer pairs Mb-COIF121 and Mb-COIR280 for *Savage Harvester Ant* was evaluated by agarose gel electrophoresis, using PCR reaction conditions (ng / μL) and primers. To ensure the reliability of the results, each experiment was repeated at least three times.

[0071] The results are as follows Figure 2 As shown.

[0072] Strong detection signals were amplified when the template DNA concentration was between 0.1 ng / μL and 10 ng / μL. For a template DNA concentration of 0.01 ng / μL, the amplification product was significantly reduced, and the detection signal was weak. When the template concentration was below 0.001 ng / μL, no amplification product was found, or the detection signal was too weak to detect. This indicates that the detection limit of this method is 0.01 ng / μL, and the optimal detection concentration is 0.1–10 ng / μL.

[0073] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A primer pair for identifying barbaric harvester ants, characterized in that, It consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:

6.

2. A reagent for identifying barbaric harvester ants, characterized in that, Includes the primer pair as described in claim 1.

3. A reagent kit for identifying barbaric harvester ants, characterized in that, Includes the primer pair as described in claim 1 or the reagent as described in claim 2.

4. The reagent kit according to claim 3, characterized in that, The kit also includes a PCR amplification premix.

5. The reagent kit according to claim 4, characterized in that, The PCR amplification premix includes at least one of a stabilizer, a DNA polymerase, Mg 2+ and dNTPs.

6. The use of the primer pair of claim 1, the reagent of claim 2, or the kit of any one of claims 3 to 5 in any one of A1) to A6): A1) Distinguish between the Savage Harvester Ant and its closely related species; A2) Prepare products that distinguish between Savage Harvester Ant and its closely related species; A3) Identification or auxiliary identification of Savage Harvester Ants; A4) Prepare products for identification or to assist in the identification of Savage Harvester Ants; A5) To identify or assist in identifying whether the sample to be tested contains Savage Harvester Ants; A6) Prepare products for identification or auxiliary identification of whether the sample to be tested contains Savage Harvester Ants; The closely related species include at least one of the following: Bighead Harvester Ant, Bryce Harvester Ant, White-spined Harvester Ant, Craftsman Harvester Ant, Oriental Harvester Ant, Needle-haired Harvester Ant, Strong Harvester Ant, Industrious Harvester Ant, and Ebony Harvester Ant.

7. A method for detecting or assisting in the detection of whether a sample to be tested is a Savage Harvester Ant or whether it contains Savage Harvester Ants, characterized in that, Includes the following steps: The genomic DNA of the sample to be tested is amplified by PCR using the primers of claim 1, the reagents of claim 2, or the kits of any one of claims 3 to 5 to obtain amplification products; the amplification products are detected to assess whether the sample to be tested is a Savage Harvester Ant or whether it contains Savage Harvester Ant.

8. The method according to claim 7, characterized in that, The annealing temperature for the PCR amplification was 52℃-62℃.

9. The method according to claim 7, characterized in that, The PCR amplification reaction conditions are as follows: denaturation at 94-96℃ for 30s-1 min; denaturation at 94-98℃ for 5-30s, annealing at 60-62℃ for 5-10s, extension at 68-72℃ for 10-30s, for 25-35 cycles; and total extension at 68-72℃ for 3-5 min.

10. The method according to claim 7, characterized in that, The criteria for evaluating whether the test sample is a Savage Harvester Ant or contains Savage Harvester Ant include: if the fragment size of the amplification product is 159-161 bp, then the test sample is a Savage Harvester Ant or contains Savage Harvester Ant; otherwise, the test sample is not a Savage Harvester Ant or does not contain Savage Harvester Ant.

11. A system for detecting barbaric harvester ants, characterized in that: The system includes: An amplification module is used to perform PCR amplification on the genomic DNA of a sample to be tested using the primer pair described in claim 1, the reagent described in claim 2, or the kit described in any one of claims 3 to 5, to obtain amplification products; The result evaluation module is used to detect the amplification product and evaluate whether the sample to be tested is a Savage Harvester Ant or whether it contains Savage Harvester Ants.

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