Primer probe combination and kit for identifying solenopsis invicta and RPA-LFD method for identifying solenopsis invicta

Abstract

The invention relates to the technical field of biological detection, in particular to a primer probe combination and a kit for identifying solenopsis invicta and an RPA-LFD method for identifying the solenopsis invicta. The invention provides a primer probe combination for identifying solenopsis invicta, and the primer probe combination has good specificity and can effectively identify the solenopsis invicta. The invention further provides an RPA-LFD method for identifying the solenopsis invicta based on the primer probe combination, and the method is good in specificity, high in sensitivity, low in reaction condition requirement, easy to operate, independent of complex equipment and capable of rapidly conducting detection outdoors.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a primer-probe combination, kit, and RPA-LFD method for identifying red imported fire ants. Background Technology

[0002] Red imported fire ants are omnivorous insects that can damage more than 50 kinds of crops. They feed on crop seeds, tender stems, buds, roots, and fruits, leading to reduced germination rates for some seeds. Furthermore, red imported fire ants attack poultry and livestock, increasing disease incidence and reducing production efficiency, making them a seriously dangerous invasive species. Therefore, the control of red imported fire ants is crucial. Due to their small size and morphological similarity to other ant species, accurate identification during field surveys is difficult. Molecular detection is a reliable method for identifying biological species. PCR or qPCR are fundamental and widely used molecular detection techniques, but PCR requires complex reaction systems, long detection times, and sophisticated temperature-controlled equipment. Traditional nucleic acid extraction methods, such as column extraction or magnetic bead extraction, are cumbersome, complex, time-consuming, and require specialized personnel. QPCR testing, which combines traditional nucleic acid extraction with PCR testing technology, is cumbersome, time-consuming, requires sophisticated instruments and professional personnel, and cannot achieve rapid, real-time testing or be completed in the field, thus limiting its use.

[0003] Recombinase polymerase amplification (RPA) is a novel isothermal nucleic acid amplification technique that has developed rapidly in recent years and is increasingly being used. It is a highly sensitive technique that can rapidly amplify DNA at a relatively low constant temperature (40±2℃), shortening the isothermal amplification reaction time to 10–15 minutes. RPA-LFD technology, combining RPA with lateral flow dipsticks (LFD), enables rapid and simple visual detection of nucleic acids. Using RPA-LFD technology, it is expected to rapidly identify suspected red imported fire ants in fields and other outdoor areas. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a primer-probe combination, kit, and RPA-LFD method for identifying red imported fire ants, aiming to solve the technical problem that traditional PCR or qPCR is not suitable for rapid identification of suspected red imported fire ants in the field or in the wild.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a primer-probe combination for identifying red imported fire ants, the primer-probe combination comprising an F1 primer, an R1 primer, and a P1 probe; F1 primer: 5'-Biotin-CGTCTACGACGACGACGACGCTCTCAACA-3'; R1 primer: 5'-TTCAACGAATCTCGAGTAGAGTGGTGCCGCACC-3'; P1 probe sequence: 5'-CTTCACTTAGATTAGGGAATATCCTTCAGTCATACAACGTTAATAAAT-3'; The T base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end.

[0006] The primer-probe combination for identifying red imported fire ants, wherein the fluorescent group is FAM, TET, HEX or CY3.

[0007] A second aspect of the present invention provides an RPA-LFD kit for identifying red imported fire ants, the kit comprising the primer-probe combination as described above.

[0008] The RPA-LFD kit for identifying red imported fire ants further includes: amplification lyophilized powder, activator, and LFD test strips.

[0009] The RPA-LFD kit for identifying red imported fire ants further includes: a mixed enzyme solution, a buffer, an activator, and an LFD test strip.

[0010] The RPA-LFD kit for identifying red imported fire ants includes a mixed enzyme solution comprising the following components: 10–30 ng / μL of Bsu DNA polymerase, 300–500 ng / μL of SSB protein, 50–90 ng / μL of UvsX recombinase, 5–20 ng / μL of UvsY protein, 40–60 ng / μL of creatine kinase, and 0.5–1 U / μL of NFO enzyme.

[0011] A third aspect of this invention provides an RPA-LFD method for identifying red imported fire ants, comprising the following steps: S01. Obtain the nucleic acid of the target object; S02. RPA reaction is performed using the nucleic acid of the target to be identified and the primer and probe combination as described above to obtain the amplification product; S03. Dilute the amplification product and add it to the LFD test strip for detection to obtain the identification results.

[0012] The RPA-LFD method for identifying red imported fire ants, wherein obtaining the nucleic acid of the target ant includes the following steps: Take a centrifuge tube, put the suspected red imported fire ant sample into the centrifuge tube, add lysis buffer, grind the body of the ant for 2-3 minutes to make the body into fine particles, and let stand for 1-2 minutes. Aspirate the liquid from the middle of the centrifuge tube and add it to a container containing nucleic acid diluent. Mix well to obtain the nucleic acid extract.

[0013] The RPA-LFD method for identifying red imported fire ants, wherein the RPA reaction is performed using the nucleic acid of the target and the primer-probe combination as described above to obtain the amplification product, includes the following steps: The primer-probe combination and activator are mixed and diluted to obtain the reaction agent; Add nucleic acid extraction solution to the reaction tube containing the amplification lyophilized powder, then add the reaction reagent and mix well; The amplification product was obtained by incubating at 37–42℃ for 12–15 min.

[0014] The RPA-LFD method for identifying red imported fire ants, wherein the step of diluting the amplification product and adding it to an LFD test strip for detection to obtain the identification result includes the following steps: Aspirate the amplification product and add it to a container containing the product diluent. Mix well to obtain the amplification product diluent. Add the diluted amplified product to the LFD test strip to obtain the identification results.

[0015] Beneficial effects: This invention provides a primer-probe combination for identifying red imported fire ants. The primer-probe combination is used for identifying red imported fire ants by the RPA-LFD method and has the advantages of high accuracy and high sensitivity.

[0016] This invention also provides an RPA-LFD method for identifying red imported fire ants. The method uses the primer-probe combination described above. During detection, the reaction conditions are low, the operation is simple, and the detection time is short. It can be applied to the identification of red imported fire ants in field or wild environments. Attached Figure Description

[0017] Figure 1 This is the specific detection result of Example 1.

[0018] Figure 2 The results are the sensitivity test results for Example 1.

[0019] Figure 3This is the specific detection result of Example 2.

[0020] Figure 4 This is the specific detection result for Comparative Example 1. Detailed Implementation

[0021] This invention provides a primer-probe combination, a kit, and an RPA-LFD method for identifying red imported fire ants. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0022] This invention provides a primer-probe combination for identifying red imported fire ants, the primer-probe combination comprising an F1 primer, an R1 primer, and a P1 probe; F1 primer: 5'-Biotin-CGTCTACGACGACGACGACGCTCTCAACA-3', whose nucleotide sequence is SEQ ID NO: 1; R1 primer: 5'-TTCAACGAATCTCGAGTAGAGTGGTGCCGCACC-3', whose nucleotide sequence is SEQ ID NO: 2; P1 probe sequence: 5'-CTTCACTTAGATTAGGGAATATCCTTCAGTCATACAACGTTAATAAAT-3', whose nucleotide sequence is SEQ ID NO: 3; The T base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end. This primer-probe combination exhibits good specificity in detecting red imported fire ants.

[0023] Specifically, the fluorescent group can be FAM, TET, HEX or CY3.

[0024] The present invention also provides an RPA-LFD kit for identifying red imported fire ants, the kit comprising the primer-probe combination as described above.

[0025] In one embodiment, the kit includes, in addition to the primer and probe combination, amplification lyophilized powder, activator, and LFD test strips.

[0026] In another embodiment, the kit, in addition to containing a primer-probe combination, also includes: a mixed enzyme solution, a buffer, an activator, and an LFD test strip.

[0027] Preferably, the mixed enzyme solution comprises the following components: 10–30 ng / μL of Bsu DNA polymerase, 300–500 ng / μL of SSB protein, 50–90 ng / μL of UvsX recombinase, 5–20 ng / μL of UvsY protein, 40–60 ng / μL of creatine kinase, and 0.5–1 U / μL of NFO enzyme.

[0028] Preferably, the kit may further include a nucleic acid extraction composition; the nucleic acid extraction composition includes a lysis buffer and a nucleic acid diluent.

[0029] The lysis buffer comprises: 0.15–0.3 M NaOH, 0.05–0.1 M Tris, pH = 12.0–12.5, and 1–5 mM EDTA.

[0030] The nucleic acid diluent is 10–50 mM Tris-H, pH = 7.00–8.00.

[0031] This invention also provides an RPA-LFD method for identifying red imported fire ants, comprising the following steps: S01. Obtain the nucleic acid of the target object; S02. RPA reaction is performed using the nucleic acid of the target to be identified and the primer and probe combination as described above to obtain the amplification product; S03. Dilute the amplification product and add it to the LFD test strip for detection to obtain the identification results.

[0032] Preferably, obtaining the nucleic acid of the object to be identified includes the following steps: Take a 1.5 mL centrifuge tube, put the suspected red imported fire ant sample into the centrifuge tube, add 300-400 μL of lysis buffer to each sample, grind the body of the test ant for 2-3 min to make the body into fine particles, and let stand for 1-2 min. Aspirate the liquid from the middle of the centrifuge tube and add it to a container containing nucleic acid diluent. Mix well to obtain the nucleic acid extract.

[0033] Preferably, the step of performing an RPA reaction using the nucleic acid of the target to be identified and the primer-probe combination as described above to obtain the amplification product includes the following steps: The primer-probe combination and activator are mixed and diluted to obtain the reaction agent; Add nucleic acid extraction solution to the reaction tube containing the amplification lyophilized powder, then add the reaction reagent and mix well; Incubate at 37–42℃ for 12–15 minutes to obtain the amplification product. 37–42℃ is the optimal temperature range. In the field, the temperature can be approached by firmly holding the reaction tube in your hand.

[0034] Specifically, the amplification lyophilized powder includes a buffer system, amplification-related enzymes, and dNTPs.

[0035] Preferably, the step of diluting the amplification product and adding it to the LFD test strip for detection to obtain the identification result includes the following steps: Aspirate the amplification product and add it to a container containing the product diluent. Mix well to obtain the amplification product diluent. Add the diluted amplified product to the LFD test strip to obtain the identification results.

[0036] Example 1 A primer-probe combination for identifying red imported fire ants, the primer-probe combination comprising an F1 primer, an R1 primer, and a P1 probe; F1 primer: 5'-Biotin-CGTCTACGACGACGACGACGCTCTCAACA-3'; R1 primer: 5'-TTCAACGAATCTCGAGTAGAGTGGTGCCGCACC-3'; P1 probe sequence: 5'-CTTCACTTAGATTAGGGAATATCCTTCAGTCATACAACGTTAATAAAT-3'; The T base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end; An RPA-LFD method for identifying red imported fire ants includes the following steps: S01. Nucleic acid extraction Take a new 1.5 mL centrifuge tube, and use tweezers to place 6 to 10 ant samples into the centrifuge tube. Use a 100 μL double-capsule pipette for each sample, add 300 to 400 μL of lysis buffer, and grind the ant body with a grinding pestle for 2 to 3 minutes to make the body into fine particles. Let it stand for 1 to 2 minutes. The lysis buffer formulation is: 0.1M NaOH, 0.05M Tris, pH=12.0~12.5, 1mM EDTA; Use a 50μL double-ended pipette to draw 50μL of the liquid from the middle of the centrifuge tube, and then add it to dropper bottle No. 1 containing 1000μL of nucleic acid diluent. Tighten the cap, shake well by inverting the bottle, and obtain the nucleic acid extract. The formula for the nucleic acid dilution buffer is: 10–50 mM Tris-H, pH = 7.00–8.00; SO2.RPA reaction Using commercially available RPA reagents as raw materials, the reagent kit includes amplification lyophilized powder (containing amplification-related enzymes and buffers) and activator. After dissolving the activator at room temperature, pipette and mix well. Pipette 60-65 μL of activator, 40-45 μL each of F1 and R1 primers, and 10-15 μL of P1 probe into an empty dropper bottle No. 2. Then, add 530-550 μL of nucleic acid dilution buffer to dropper bottle No. 2, making the total liquid volume 700 μL. Cap dropper bottle No. 2 and shake it until the liquid is homogeneous to obtain the reaction reagent. Take out the reaction tube containing the amplification lyophilized powder, open the cap, and use the No. 1 dropper bottle containing nucleic acid extraction solution. After opening the cap, first drop 2 drops into the waste liquid container, and then drop 1 drop into the amplification lyophilized powder. Take out dropper bottle No. 2, open the cap and drop 2 drops into the waste liquid bucket, then drop 1 drop of the reagent into the amplification lyophilized powder. Use a pipette to carefully blow and stir the liquid formed after the amplification lyophilized powder melts, blowing and stirring 15 to 20 times until completely uniform. Incubate at 37–42℃ for 12–15 min to obtain the amplification product; In the RPA reaction, the commercially available RPA reagent is specifically the chromatography-type RPA (NFO probe) (in situ lyophilized powder) produced by Yisheng Biotechnology (Shanghai) Co., Ltd. S03. Test strip detection After RPA incubation, carefully open the cap and use a 20μL double-ended pipette to draw 20μL of amplification product. Add the product to a dropper containing 780μL of product diluent. Tighten the cap and shake well by inverting the bottle. The product diluent is formulated as follows: 20-50mM Tris-H, pH=7.00-8.00. After shaking well, open the cap, drop 2 drops into the waste liquid container, then drop 3 drops into the LFD test strip card, wait 2-3 minutes, and observe the results. The results displayed within 1-5 minutes shall be taken as the standard. Results displayed after 5 minutes shall not be used as the basis for judging positive or negative. Result interpretation: If there are lines at both C and T on the reagent card (including blurry lines), the result is positive; if there is only a line at C, the result is negative; if there is no line at C (regardless of whether there is a line at T), the reagent card is invalid and needs to be replaced for testing.

[0037] Specific detection Five species of fire ants closely related to the red imported fire ant (tropical fire ant, wood fire ant, black fire ant, Brazilian fire ant, and cotton fire ant), along with collected small fire ants, broad-headed fire ants, Japanese carpenter ants, and red imported fire ants, totaling nine species, were used as test samples. The tests were conducted according to the method in Example 1, with three negative controls (nuclease-free water) set up to avoid false positives. The results are as follows: Figure 1 As shown in Table 1. Figure 1 The corresponding sample information table for the results.

[0038] Table 1

[0039] from Figure 1 The results show that only red imported fire ants showed positive results, while all other samples and negative controls showed negative results. This indicates that the primer-probe combination in this embodiment has good specificity and can accurately identify red imported fire ants from fire ants and other ants with similar homology.

[0040] Sensitivity test Nucleic acid extracts from red imported fire ant samples were obtained according to the nucleic acid extraction method described in Example 1, and further diluted 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 times to obtain a total of 11 different concentrations of DNA templates. The samples were then tested according to the identification method described in Example 1, and the results are as follows: Figure 2 As shown.

[0041] from Figure 2 As can be seen, even after the original nucleic acid template was diluted 256 times, there were still relatively obvious bands during detection. However, the bands were not obvious after the template was diluted 512 times, and there were no bands at all after a dilution of 1024 times. Based on the dilution calculation, it can be theoretically shown that even a sample of 0.04 red imported fire ants can be detected, indicating that this method has extremely high sensitivity.

[0042] Example 2 A primer-probe combination for identifying red imported fire ants, the primer-probe combination comprising an F1 primer, an R1 primer, and a P1 probe; F1 primer: 5'-Biotin-CGTCTACGACGACGACGACGCTCTCAACA-3'; R1 primer: 5'-TTCAACGAATCTCGAGTAGAGTGGTGCCGCACC-3'; P1 probe sequence: 5'-CTTCACTTAGATTAGGGAATATCCTTCAGTCATACAACGTTAATAAAT-3'; The T base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end; An RPA-LFD method for identifying red imported fire ants includes the following steps: S01. Nucleic acid extraction Take a new 1.5 mL centrifuge tube, and use tweezers to place 6 to 10 ant samples into the centrifuge tube. Use a 100 μL double-capsule pipette for each sample, add 300 to 400 μL of lysis buffer, and grind the ant body with a grinding pestle for 2 to 3 minutes to make the body into fine particles. Let it stand for 1 to 2 minutes. The lysis buffer formulation is: 0.1M NaOH, 0.05M Tris, pH=12.0~12.5, 1mM EDTA; In a 1.5 mL centrifuge tube, use a 1000 μL double-ended capillary pipette to draw 1000 μL of nucleic acid diluent and add it to the 1.5 mL centrifuge tube; insert a 50 μL double-ended capillary pipette into the centrifuge tube containing the sample, draw 50 μL of the intermediate liquid and add it to the 1.5 mL centrifuge tube containing 1000 μL of nucleic acid diluent, tighten the cap, and shake well up and down to obtain the nucleic acid extract; The formula for the nucleic acid dilution buffer is: 10–50 mM Tris-H, pH = 7.00–8.00; SO2.RPA reaction Using commercially available RPA reagents as raw materials, the reagent kit includes amplification lyophilized powder (containing amplification-related enzymes and buffer) and activator. After dissolving the activator at room temperature, pipette and mix well. Pipette 60-65 μL of activator, 40-45 μL each of F1 and R1 primers, and 10-15 μL of P2 probe into an empty 1.5 mL centrifuge tube. Then, add 530-550 μL of nucleic acid dilution buffer into the 1.5 mL centrifuge tube, for a total liquid volume of 700 μL, to obtain the reaction reagent. Take out the reaction tube containing the amplification lyophilized powder, open the cap, use a 20μL double-ended pipette to draw 20μL of nucleic acid extraction solution, and add it to the amplification lyophilized powder; Take out a 1.5mL centrifuge tube containing the reagent, use a 20μL double-ended pipette to draw 20μL of the reagent, and add it dropwise to the amplification lyophilized powder. Use a pipette to carefully blow the liquid formed after the amplification lyophilized powder melts, blowing 15 to 20 times until completely homogeneous. Incubate at 37–42℃ for 12–15 min to obtain the amplification product; The commercially available RPA reagent is specifically the chromatography-type RPA (NFO probe) (in-situ lyophilized powder) produced by Yisheng Biotechnology (Shanghai) Co., Ltd. S03. Test strip detection After RPA incubation, carefully open the cap and use a 20μL double-ended pipette to aspirate 20μL of amplification product. Add the product to a 1.5mL centrifuge tube containing 780μL of product diluent. Tighten the cap and shake well by inverting the tube. The product diluent is formulated as follows: 20-50mM Tris-H, pH=7.00-8.00. After shaking well, open the cap and use a 50μL double-ended pipette to draw 50μL of amplification product dilution solution and drop it onto the test strip. Repeat once, for a total of 100μL of amplification product dilution solution to be dropped onto the LFD test strip. Wait 2-3 minutes and observe the results. The results displayed within 1-5 minutes are considered accurate. Results displayed after 5 minutes are not considered as a basis for judging positive or negative results.

[0043] In this embodiment, a 1.5mL centrifuge tube is used instead of the dropper bottle used in Example 1, and the detection operation can be completed in the same way.

[0044] Specific detection Red imported fire ants, tropical fire ants, wood fire ants, and black fire ants were used as test samples, and the tests were performed according to the method in Example 2. A blank control (nuclease-free water) was also included to avoid false positives. The results are as follows: Figure 3 As shown in Table 2. Figure 3 The corresponding sample information table for the results.

[0045] Table 2

[0046] from Figure 3 The results show that the detection method in Example 2 can effectively detect red imported fire ants and is specific to similar species such as tropical fire ants, wood fire ants, and black fire ants, demonstrating good detection effectiveness and specificity.

[0047] Comparative Example 1 A primer-probe combination comprising an F2 primer, an R2 primer, and a P2 probe; F2 primer: 5'-Biotin-CGAGCATCCGAGTGAACGATCCTCCTAGC-3', whose nucleotide sequence is SEQ ID NO: 4; R2 primer: 5'-GCTTCTATGTGCGGCCATACCATTTAGCCTTC-3', whose nucleotide sequence is SEQ ID NO: 5; P2 probe sequence: 5'-ATAGCATTACCATACGTCGCGCAGTGGAATCCGCACGGGTCTTCAA-3', whose nucleotide sequence is SEQ ID NO: 6; The A base of the P2 probe is replaced by dSpacer at a position 29 bp from the 5' end; the P2 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end; Specific detection Red imported fire ants, tropical fire ants, wood fire ants, and black imported fire ants were used as test samples. A comparative primer-probe combination was employed, and the detection was performed according to the method in Example 1. A blank control (nuclease-free water) was also included to avoid false positives. The results are as follows: Figure 3 As shown in Table 3. Figure 4 The corresponding sample information table for the results.

[0048] Table 3

[0049] from Figure 4 The results showed that the primer-probe combination in the comparative ratio could not effectively detect red imported fire ants.

[0050] Example 3 An RPA-LFD method for identifying red imported fire ants, which differs from Example 1 in that... In S01, 450 μL of nucleic acid diluent was added to a 1.5 mL centrifuge tube; 50 μL of the intermediate liquid was added to the 1.5 mL centrifuge tube containing 450 μL of nucleic acid diluent, the cap was tightened, and the tube was shaken upside down to mix well to obtain nucleic acid extract (template DNA). In S02, instead of using amplification lyophilized powder, a mixed enzyme solution and its buffer are used. The mixed enzyme solution contains the following components: 20 ng / μL of Bsu DNA polymerase, 400 ng / μL of SSB protein, 60 ng / μL of UvsX recombinase, 10 ng / μL of UvsY protein, 50 ng / μL of creatine kinase, and 0.5 U / μL of NFO enzyme. The RPA reaction steps are as follows: (1) 10 minutes in advance, place the buffer (2×) (reaction buffer) and activator (350mM magnesium acetate) at room temperature to dissolve completely. After the reagents are dissolved, gently shake to mix for 3-5 seconds, centrifuge for 2-3 seconds, and add the reagents to the reaction tube in the order shown in Table 4.

[0051] Table 4

[0052] The buffer (2×) consists of: 100 mM Tris-HCl solution at pH 7.9, 200 mM potassium acetate, 400 μM dNTPs, 4 mM dithiothreitol, 100 mM creatine phosphate, and 6 mM ATP. (2) After adding the above components, add 2 μL of activator to each tube cap, carefully put the cap on, and then centrifuge for 2-3 seconds to allow the activator to enter the reaction mixture. Immediately invert the tube 8-10 times or shake it to mix for 3-5 seconds, and then immediately centrifuge for 2-3 seconds to allow all the reagents to sink to the bottom of the tube. (3) Immediately add the reaction tube to a metal bath (40±2℃) and incubate for 15 min.

[0053] After shaking well, open the cap and use a 50μL double-ended pipette to draw 50μL of amplification product dilution solution and drop it onto the test strip. Repeat once, for a total of 100μL of amplification product dilution solution to be dropped onto the LFD test strip. Wait 2-3 minutes and observe the results. The results displayed within 1-5 minutes are considered accurate. Results displayed after 5 minutes are not considered as a basis for judging positive or negative results.

[0054] Sensitivity test Nucleic acid extracts from red imported fire ant samples were obtained according to the nucleic acid extraction method in Example 3, and then further diluted by 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 times to obtain a total of 11 DNA templates with different concentrations. The samples were then tested according to the identification method in Example 3, and the results are shown in Table 5.

[0055] Table 5

[0056] In Table 5, "detected" means there is a distinct band, and "not detected" means there is no distinct band.

[0057] Table 5 Results and Figure 2 Similarly, after the original nucleic acid template was diluted 256 times, a relatively obvious band was still visible during detection, which was considered as detection. However, after the template was diluted 512 times, the band was not obvious and was considered as not detected. After a dilution of 1024 times, there was no band at all. Based on the dilution calculation, it can be theoretically stated that even a sample of 0.04 red imported fire ants can be detected, indicating that this method also has extremely high sensitivity.

[0058] Comparative Example 2 An RPA-LFD method for identifying red imported fire ants differs from Example 3 in that the formulation of the mixed enzyme solution is different. In Comparative Example 2, the concentration of UvsX recombinant enzyme in the mixed enzyme solution is 20 ng / μL.

[0059] Sensitivity test Nucleic acid extracts from red imported fire ant samples were obtained according to the nucleic acid extraction method described in Example 3, and then further diluted by 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 times to obtain a total of 11 DNA templates of different concentrations. The samples were then tested according to the identification method described in Example 3, and the results are shown in Table 6.

[0060] Table 6

[0061] As can be seen from the results in Table 6, when the concentration of UvsX recombinase in the mixed enzyme solution is too low, it will lead to a decrease in the sensitivity of the detection.

[0062] Comparative Example 3 An RPA-LFD method for identifying red imported fire ants differs from Example 3 in that the formulation of the mixed enzyme solution is different. In Comparative Example 3, the SSB protein concentration of the mixed enzyme solution is 100 ng / μL.

[0063] Sensitivity test Nucleic acid extracts from red imported fire ant samples were obtained according to the nucleic acid extraction method in Example 3, and then further diluted by 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 times to obtain a total of 11 DNA templates with different concentrations. The samples were then tested according to the identification method in Example 3, and the results are shown in Table 7.

[0064] Table 7

[0065] As can be seen from the results in Table 7, when the concentration of SSB protein in the mixed enzyme solution is too low, it will also lead to a decrease in the sensitivity of the detection.

[0066] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solution and inventive concept of the present invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A primer-probe combination for identifying red imported fire ants, characterized in that, The primer-probe combination includes an F1 primer, an R1 primer, and a P1 probe; F1 primer: 5'-Biotin-CGTCTACGACGACGACGACGCTCTCAACA-3'; R1 primer: 5'-TTCAACGAATCTCGAGTAGAGTGGTGCCGCACC-3'; P1 probe sequence: 5'-CTTCACTTAGATTAGGGAATATCCTTCAGTCATACAACGTTAATAAAT-3'; The T base of the P1 probe is replaced by dSpacer at a position 30 bp from the 5' end; the P1 probe is attached to a fluorescent group at the 5' end and to ddC at the 3' end.

2. The primer-probe combination for identifying red imported fire ants according to claim 1, characterized in that, The fluorescent group is FAM, TET, HEX or CY3.

3. An RPA-LFD kit for identifying red imported fire ants, characterized in that, The kit includes the primer-probe combination as described in claim 1 or 2.

4. The RPA-LFD kit for identifying red imported fire ants according to claim 3, characterized in that, The kit also includes: amplification lyophilized powder, activator, and LFD test strips.

5. The RPA-LFD kit for identifying red imported fire ants according to claim 3, characterized in that, The kit also includes: mixed enzyme solution, buffer, activator, and LFD test strips.

6. The RPA-LFD kit for identifying red imported fire ants according to claim 5, characterized in that, The mixed enzyme solution comprises the following components: 10–30 ng / μL of Bsu DNA polymerase, 300–500 ng / μL of SSB protein, 50–90 ng / μL of UvsX recombinase, 5–20 ng / μL of UvsY protein, 40–60 ng / μL of creatine kinase, and 0.5–1 U / μL of NFO enzyme.

7. An RPA-LFD method for identifying red imported fire ants, characterized in that, Includes the following steps: S01. Obtain the nucleic acid of the target object; S02. Using the nucleic acid of the target to be identified and the primer-probe combination as described in claim 1 or 2, an RPA reaction is performed to obtain the amplification product; S03. Dilute the amplification product and add it to the LFD test strip for detection to obtain the identification results.

8. The RPA-LFD method for identifying red imported fire ants according to claim 7, characterized in that, The process of obtaining the nucleic acid of the object to be identified includes the following steps: Take a centrifuge tube, put the suspected red imported fire ant sample into the centrifuge tube, add lysis buffer, grind the body of the ant for 2-3 minutes to make the body into fine particles, and let stand for 1-2 minutes. Aspirate the liquid from the middle of the centrifuge tube and add it to a container containing nucleic acid diluent. Mix well to obtain the nucleic acid extract.

9. The RPA-LFD method for identifying red imported fire ants according to claim 8, characterized in that, The step of performing an RPA reaction using the nucleic acid of the target object and the primer-probe combination as described in claim 1 or 2 to obtain the amplification product includes the following steps: The primer-probe combination and activator are mixed and diluted to obtain the reaction agent; Add nucleic acid extraction solution to the reaction tube containing the amplification lyophilized powder, then add the reaction reagent and mix well; The amplification product was obtained by incubating at 37–42℃ for 12–15 min.

10. The RPA-LFD method for identifying red imported fire ants according to claim 9, characterized in that, The process of diluting the amplification product and adding it to an LFD test strip for detection to obtain identification results includes the following steps: Aspirate the amplification product and add it to a container containing the product diluent. Mix well to obtain the amplification product diluent. Add the diluted amplified product to the LFD test strip to obtain the identification results.

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