Primer probe combination, kit and method for identifying Solenopsis invicta through fluorescent RPA (recombinase polymerase amplification)

Abstract

The invention relates to the technical field of biological detection, in particular to a primer probe combination, a kit and a method for identifying Solenopsis invicta through fluorescent RPA (recombinase polymerase amplification). The primer probe combination for identifying the Solenopsis invicta through the fluorescent RPA comprises a primer F1, a primer R1 and a probe P1. Wherein the nucleotide sequence of the probe P1 is SEQ ID NO: 3, and thymine at the position, 27bp away from the 5'end, of the probe P1 is modified by a fluorophore FAM; a basic group at the position, 28bp away from the 5'end, of the probe P1 is replaced by dSpacer, thymine at the position, 30bp away from the 5 'end, of the probe P1 is modified by a quenching group BHQ1, and the 3' end of the probe P1 is connected with ddC. The primer probe combination is used for identifying the Solenopsis invicta through fluorescent RPA and has the advantages of being good in specificity and high in sensitivity.

Description

Technical Field

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

[0002] Like red imported fire ants, small fire ants are among my country's invasive species. They can spread rapidly through covert means such as soil, seedlings, and cargo transportation. Once they invade a new area, they use their large colony size to prey on native insects and small vertebrates (such as lizards and frogs), and can even inject formic acid-containing venom through their stings, causing physiological damage to native species. Accurate identification is the "first line of defense" against their invasion. If they can be accurately identified in the early stages of invasion, timely extermination measures (such as the application of fipronil pesticides and the establishment of bait stations) can be initiated to prevent them from forming stable populations.

[0003] Recombinase polymerase amplification (RPA) is a novel isothermal nucleic acid amplification technology that has attracted widespread attention in recent years. Leveraging its unique advantages, it has been rapidly promoted and applied in multiple fields. The core competitiveness of this technology lies in its ability to efficiently complete the amplification reaction in a mild, isothermal environment of 40±2℃, without the need for complex temperature cycling equipment. This characteristic significantly reduces the requirements for experimental environment and instruments, making it highly adaptable to scenarios such as grassroots laboratory testing and rapid on-site screening, effectively filling the application gap of traditional nucleic acid amplification technologies that are highly dependent on specialized equipment. Fluorescent RPA technology, developed based on RPA, not only fully retains the core advantages of "mild isothermal and low instrument dependence," but also achieves significant breakthroughs in detection performance. Its key improvement lies in the targeted design and synthesis of specific fluorescent primers and probes, allowing the nucleic acid amplification process and result detection to be carried out simultaneously: the entire detection process, from initiation to result output, takes only 10-15 minutes. Compared to traditional nucleic acid detection methods (such as PCR), the detection cycle is significantly compressed, enabling a faster response to immediate testing needs. Summary of the Invention

[0004] The purpose of this invention is to provide a primer-probe combination, kit, and method for identifying small fire ants using fluorescent RPA, aiming to provide a primer-probe combination suitable for identifying small fire ants using fluorescent RPA, thereby increasing the means and accuracy of fire ant identification.

[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 small fire ants using fluorescent RPA, including an F1 primer, an R1 primer, and a P1 probe; F1 primer: 5'-CACAAACAGCCGCTCATGAAATTCCAACGC-3'; R1 primer: 5'-CCGCGTTCTCGCTTCTGGTTCATCCGATTG-3'; P1 probe sequence: 5'-CATCGCTGCACGTCGACGATGTTTCGTATTCGCGTCGAGTTAATGCA-3'; The thymine at a position 27 bp from the 5' end of the P1 probe is modified with the fluorescent group FAM. The A base at a position 28 bp from the 5' end of the P1 probe is replaced by dSpacer; The thymine at a position 30 bp from the 5' end of the P1 probe is modified with the quenching group BHQ1. The P1 probe is connected to ddC at its 3' end.

[0006] A second aspect of the present invention provides a fluorescent RPA kit for identifying small fire ants, the kit comprising the primer-probe combination as described above.

[0007] The fluorescent RPA kit for identifying small fire ants further includes: nucleic acid diluent, activator, and amplification lyophilized powder.

[0008] The fluorescent RPA kit for identifying small fire ants further includes a lysis buffer containing the following components: 0.2–0.3 M NaOH, 0.1–0.3 M Tris, and 1–5 mM DTT, with a pH of 12.0–12.5.

[0009] The fluorescent RPA kit for identifying small fire ants further includes: nucleic acid diluent, mixed enzyme solution, buffer, and activator.

[0010] The fluorescent RPA kit for identifying small 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 1–4 U / μL of EXO enzyme.

[0011] The fluorescent RPA kit for identifying small fire ants, wherein the nucleic acid diluent is: Tris-HCl with a concentration of 10-50 mM and a pH of 7.0-8.0.

[0012] A third aspect of this invention provides a method for identifying small fire ants using fluorescent RPA, comprising the following steps: S01. Obtain the nucleic acid of the target object; S02. After mixing the nucleic acid of the target object, the amplification lyophilized powder, the activator, and the primer and probe combination described above, the mixture is placed in a fluorescence detector for reaction to obtain the detection results.

[0013] The method for identifying small fire ants using fluorescent RPA, wherein obtaining the nucleic acid of the target ant includes the following steps: Take a centrifuge tube, put the suspected small fire ant sample into the centrifuge tube, add lysis buffer, grind the body of the test 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.

[0014] The method for identifying small fire ants using fluorescent RPA, wherein the steps of mixing the nucleic acid of the target, the lyophilized amplification powder, the activator, and the primer-probe combination described above, and then placing the mixture in a fluorescence detector for reaction to obtain detection results, include the following steps: The primer-probe combination and activator are mixed and diluted to obtain the reaction agent; Add nucleic acid extraction solution to a reaction tube containing amplification lyophilized powder, then add reaction reagent, mix well, and obtain a mixture; Transfer the mixture to a detection tube, then place the detection tube into a fluorescence detector. After the fluorescence detector has finished reacting, read the detection result from the fluorescence detector.

[0015] Beneficial Effects: This invention discloses a primer-probe combination for identifying small red fire ants using fluorescent recombinase polymerase amplification (RPA) technology. This primer-probe combination is designed specifically for the nucleic acid sequence characteristics of small red fire ants, enabling precise matching of the target detection fragment and exhibiting excellent specificity in the fluorescent RPA detection system. It also possesses high sensitivity, stably achieving amplification and detection even with low concentrations of small red fire ant nucleic acid samples, meeting the identification needs of trace samples.

[0016] The fluorescent RPA method for identifying small fire ants based on the above primer-probe combination is simple to operate, has low requirements for instruments and equipment, short detection time, and high accuracy. It is suitable for rapid identification of small fire ants in grassroots or field environments. Detailed Implementation

[0017] The first aspect of the present invention provides a primer-probe combination for identifying small fire ants using fluorescent RPA, including an F1 primer, an R1 primer, and a P1 probe; 5'-CACAAACAGCCGCTCATGAAATTCCAACGC-3', whose nucleotide sequence is SEQ ID NO: 1; R1 primer: 5'-CCGCGTTCTCGCTTCTGGTTCATCCGATTG-3', whose nucleotide sequence is SEQ ID NO: 2; P1 probe sequence: 5'-CATCGCTGCACGTCGACGATGTTTCGTATTCGCGTCGAGTTAATGCA-3', whose nucleotide sequence is SEQ ID NO: 3; The thymine at a position 27 bp from the 5' end of the P1 probe is modified with the fluorescent group FAM. The A base at a position 28 bp from the 5' end of the P1 probe is replaced by dSpacer; The thymine at a position 30 bp from the 5' end of the P1 probe is modified with the quenching group BHQ1. The P1 probe is connected to ddC at its 3' end.

[0018] A second aspect of the present invention provides a fluorescent RPA kit for identifying small fire ants, the kit comprising the primer-probe combination as described above.

[0019] Preferably, the fluorescent RPA kit further includes a lysis buffer containing the following components: 0.2–0.3 M NaOH, 0.1–0.3 M Tris, and 1–5 mM DTT, with a pH of 12.0–12.5.

[0020] In one embodiment, the fluorescent RPA kit for identifying small fire ants further includes nucleic acid diluent, activator, and amplification lyophilized powder. Specifically, the amplification lyophilized powder includes amplification-related enzymes and a buffer.

[0021] Preferably, the nucleic acid diluent is: Tris-HCl with a concentration of 10-50 mM and a pH of 7.0-8.0.

[0022] In another embodiment, the fluorescent RPA kit for identifying small fire ants further includes a nucleic acid diluent, a mixed enzyme solution, a buffer, and an activator. The mixed enzyme solution comprises the following components: 10–30 ng / μL of Bsu DNA polymerase, 400–600 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 1–4 U / μL of EXO enzyme.

[0023] A third aspect of this invention provides a method for identifying small fire ants using fluorescent RPA, comprising the following steps: S01. Obtain the nucleic acid of the target object; S02. After mixing the nucleic acid of the target object, the amplification lyophilized powder, the activator, and the primer and probe combination described above, the mixture is placed in a fluorescence detector for reaction to obtain the detection results.

[0024] Preferably, obtaining the nucleic acid of the object to be identified includes the following steps: Take a centrifuge tube, put the suspected small fire ant sample into the centrifuge tube, add lysis buffer, grind the body of the test 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.

[0025] Preferably, the step of mixing the nucleic acid of the target object, the lyophilized amplification powder, the activator, and the primer-probe combination described above, and then reacting them in a fluorescence detector to obtain the detection results 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 a reaction tube containing amplification lyophilized powder, then add reaction reagent, mix well, and obtain a mixture; Transfer the mixture to a detection tube, then place the detection tube into a fluorescence detector. After the fluorescence detector has finished reacting, read the detection result from the fluorescence detector.

[0026] Example 1 A method for identifying small fire ants using fluorescent RPA includes the following steps: Nucleic acid extraction: A01. Take 3 to 5 suspected small fire ants and put them into a 1.5 mL centrifuge tube. Add 100 to 200 μL of lysis buffer to each sample tube. Grind the body with a pestle for 2 to 3 minutes to make the body into fine particles. Let it stand for 1 to 2 minutes. A02. Using a 200μL pipette, pipette 380μL of nucleic acid diluent into a new 1.5mL centrifuge tube. Then, using a 200μL pipette, insert it into the centrifuge tube containing the sample and pipette 20μL of the intermediate liquid into the centrifuge tube containing 380μL of nucleic acid diluent. Tighten the cap and shake well by inverting the tube to obtain the nucleic acid extract.

[0027] RPA reaction and detection: A03. Commercially available RPA reagents were used as raw materials, specifically fluorescent RPA (Exo probe method) (in situ lyophilized powder) produced by Yisheng Biotechnology (Shanghai) Co., Ltd. The RPA reagent kit includes amplification lyophilized powder (containing amplification-related enzymes and buffers) and activator. After dissolving the activator at room temperature, the mixture was pipetted and mixed evenly. 60-65 μL of activator, 40-45 μL each of F1 and R1 primers, and 10-15 μL of P1 probe were added to an empty 1.5 mL centrifuge tube. Then, 530-550 μL of nucleic acid dilution buffer was added to the 1.5 mL centrifuge tube, for a total liquid volume of 700 μL, to obtain the reaction reagent. A04. Take out the reaction tube containing the amplification lyophilized powder, open the cap, take 25 μL of nucleic acid extraction solution and add it to the amplification lyophilized powder. Then take 25 μL of reaction reagent and add it to the amplification lyophilized powder. Use a 200 μL pipette to carefully blow the liquid formed after the amplification lyophilized powder melts. Blow 15 to 20 times until completely homogeneous. Transfer all the liquid to the detection square tube (matching mini fluorescence detector) and cover it with the rubber cap. A05. Open the cover of the mini fluorescence detector, place the detection square tubes into the holes of the mini fluorescence detector in sequence, close the instrument cover, and start the mini fluorescence detector for detection. After the detection starts, the instrument will automatically interpret the positive and negative results of the sample within 15 minutes and display them on the instrument screen. The instrument displays "+" for positive and "-" for negative.

[0028] Following the above procedures, nucleic acids were collected from red imported fire ants, tropical fire ants, wood fire ants, black fire ants, Brazilian fire ants, cotton fire ants, small fire ants, broad-headed ants, and Japanese carpenter ants for testing. Nucleic acid dilution buffer was used as a negative control to determine the specificity of the primer-probe combination (F1 primer, R1 primer, and P1 probe). The test results are shown in Table 1. Table 1

[0029] Results analysis: Among the nine ant species tested, only the small red fire ant showed a positive result, and the positive result appeared in 5 minutes and 55 seconds, indicating that the detection efficiency was high. All other samples and negative controls showed negative results, indicating that the method has good specificity and can accurately identify small red fire ants from fire ants and other ants with similar homology.

[0030] After confirming the specificity of the primer-probe combination, sensitivity testing was performed. The nucleic acid extract of small fire ants was diluted 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, and 2048 times, respectively, with the nucleic acid dilutions used as negative controls. Fluorescent RPA detection was performed according to the above steps, and the results are shown in Table 2.

[0031] Table 2

[0032] As shown in Table 2, even after a 512-fold dilution of the original nucleic acid template, positive results were still detected, with the time to positive detection approaching the instrument threshold of 15 minutes. A 1024-fold dilution of the original nucleic acid template yielded negative results. Based on the dilution calculations, theoretically only 0.04 small fire ants are needed for identification. Using a micro-spectrophotometer, the DNA concentration of the original nucleic acid solution was measured to be 10.69 nanograms per microliter. Based on a 512-fold dilution, the detection sensitivity of this invention reaches 2.09 × 10⁻⁶. -2 The nanograms per microliter indicates that the method and primer-probe combination have extremely high sensitivity.

[0033] Comparative Example 1 A fluorescent RPA method for identifying small fire ants, which differs from Example 1 in that it uses a different primer-probe combination; In this comparative example, the primer-probe combination includes: F2 primer: 5'-ACTCGCTCCGACACGGAAACTCTGTGTGTT-3', whose nucleotide sequence is SEQ ID NO: 4; R2 primer: 5'-GTGCAGCGATGACTCTCTTGTTTCCCACCT-3', whose nucleotide sequence is SEQ ID NO: 5; P2 probe sequence: 5'-AAGAAACTGGGCCTCACAAACAGCCGCTCATGAAATTCCAACGCAAG-3', whose nucleotide sequence is SEQ ID NO: 6; The thymine at a position 28 bp from the 5' end of the P2 probe is modified with the fluorescent group FAM. The C base of the P2 probe at a position 29 bp from the 5' end is replaced by dSpacer; The thymine at a position 31 bp from the 5' end of the P2 probe is modified with the quenching group BHQ1. The P2 probe is connected to ddC at its 3' end.

[0034] Following the above procedures, nucleic acids from small fire ants, tropical fire ants, wood fire ants, and black fire ants were collected and tested. Nucleic acid dilution buffer was used as a negative control to determine the specificity of the comparative primer-probe combinations (F2 primer, R2 primer, and P2 probe). The test results are shown in Table 3. Table 3

[0035] As can be seen from the results in Table 3, the primer-probe combination in the comparative example could not effectively detect small fire ants, indicating that the detection effectiveness was poor.

[0036] Example 2 A fluorescent RPA method for identifying small fire ants differs from Example 1 in that the dilution factor of the nucleic acid extract is different, and the reagents used in the RPA reaction are different. 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, 500 ng / μL of SSB protein, 60 ng / μL of UvsX recombinase, 10 ng / μL of UvsY protein, 50 ng / μL of creatine kinase, and 1 U / μL of EXO enzyme. The RPA reaction steps are as follows: (1) 10 minutes in advance, place the buffer (2×) 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 the table below.

[0037]

[0038] 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 the cap of each tube, 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) Open the cover of the mini fluorescence detector, put the detection square tube into the holes of the mini fluorescence detector in sequence, close the instrument cover, start the mini fluorescence detector for detection. After the detection starts, the instrument will automatically read the positive and negative results of the sample within 15 minutes and display them on the instrument screen. The instrument displays "+" for positive and "-" for negative.

[0039] Sensitivity test Nucleic acid extracts from small fire ant samples were obtained according to the nucleic acid extraction method in Example 2, 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 2, and the results are shown in Table 4.

[0040] Table 4

[0041] As can be seen from the results in Table 4, the RPA reaction system of Example 2 also has good sensitivity, comparable to that of Example 1.

[0042] Comparative Example 1 A method for identifying small fire ants using fluorescent RPA differs from Example 2 in that the formulation of the mixed enzyme solution is different. In this comparative example, the concentration of EXO enzyme in the mixed enzyme solution was 0.5 U / μL.

[0043] Sensitivity test Nucleic acid extracts from small fire ant samples were obtained according to the nucleic acid extraction method in Example 2, 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 2, and the results are shown in Table 5.

[0044] Table 5

[0045] As can be seen from the results in Table 5, when the concentration of EXO enzyme in the mixed enzyme solution of Comparative Example 2 is too low, it will lead to a decrease in the sensitivity of the RPA reaction system.

[0046] Comparative Example 2 A method for identifying small fire ants using fluorescent RPA differs from Example 2 in that the formulation of the mixed enzyme solution is different. In this comparative example, the concentration of SSB protein in the mixed enzyme solution was 300 ng / μL.

[0047] Sensitivity test Nucleic acid extracts from small fire ant samples were obtained according to the nucleic acid extraction method in Example 2, 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 in Example 2, and the results are shown in Table 6.

[0048] Table 6

[0049] As can be seen from the results in Table 6, when the concentration of SSB protein in the mixed enzyme solution of Comparative Example 2 is too low, it will also lead to a decrease in the sensitivity of the RPA reaction system.

[0050] 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 small fire ants using fluorescent RPA, characterized in that, This includes F1 primers, R1 primers, and P1 probes; F1 primer: 5'-CACAAACAGCCGCTCATGAAATTCCAACGC-3'; R1 primer: 5'-CCGCGTTCTCGCTTCTGGTTCATCCGATTG-3'; P1 probe sequence: 5'-CATCGCTGCACGTCGACGATGTTTCGTATTCGCGTCGAGTTAATGCA-3'; The thymine at a position 27 bp from the 5' end of the P1 probe is modified with the fluorescent group FAM. The A base at a position 28 bp from the 5' end of the P1 probe is replaced by dSpacer; The thymine at a position 30 bp from the 5' end of the P1 probe is modified with the quenching group BHQ1. The P1 probe is connected to ddC at its 3' end.

2. A fluorescent RPA kit for identifying small fire ants, characterized in that, The kit includes the primer-probe combination as described in claim 1.

3. The fluorescent RPA kit for identifying small fire ants according to claim 2, characterized in that, Also includes: Nucleic acid diluent, activator, and lyophilized amplification powder.

4. The fluorescent RPA kit for identifying small fire ants according to claim 2, characterized in that, It also includes a lysis buffer containing the following components: NaOH at a concentration of 0.2–0.3 M, Tris at a concentration of 0.1–0.3 M, and DTT at a concentration of 1–5 mM, and the pH of the lysis buffer is 12.0–12.

5.

5. The fluorescent RPA kit for identifying small fire ants according to claim 2, characterized in that, Also includes: Nucleic acid diluent, mixed enzyme solution, buffer, activator.

6. The fluorescent RPA kit for identifying small 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, 400–600 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 1–4 U / μL of EXO enzyme.

7. The fluorescent RPA kit for identifying small fire ants according to claim 3, characterized in that, The nucleic acid diluent is Tris-HCl with a concentration of 10–50 mM and a pH of 7.0–8.

0.

8. A method for identifying small fire ants using fluorescent RPA, characterized in that, Includes the following steps: S01. Obtain the nucleic acid of the target object; S02. The nucleic acid of the target to be identified, the lyophilized amplification powder, the activator, and the primer and probe combination as described in claim 1 are mixed and then placed in a fluorescence detector for reaction to obtain the detection results.

9. The method for identifying small fire ants using fluorescent RPA according to claim 8, 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 small 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.

10. The method for identifying small fire ants using fluorescent RPA according to claim 8, characterized in that, The process of mixing the nucleic acid of the target object, the lyophilized amplification powder, the activator, and the primer and probe combination as described in claim 1, and then reacting them in a fluorescence detector to obtain detection results 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 a reaction tube containing lyophilized amplification powder, then add reaction reagent, mix well, and obtain a mixture; Transfer the mixture to a detection tube, then place the detection tube into the fluorescence detector. After the fluorescence detector has finished reacting, read the detection result from the fluorescence detector.

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