LAMP (Loop-Mediated Isothermal Amplification)-based rapid detection primer group, method and kit for Solenopsis invicta

Abstract

The invention discloses a LAMP (loop-mediated isothermal amplification)-based rapid detection primer group, method and kit for Solenopsis invicta. According to the invention, by innovatively combining a commercial isothermal amplification kit, a self-designed specific primer, a sample releasing agent and a portable constant-temperature heater, rapid, portable and low-cost detection in a field environment is realized; detection can be completed within 45 min, the sensitivity reaches 2.31 * 10 <-3 > ng / L, and the specificity is larger than or equal to 99%. The method can be widely applied to the scenes of agricultural quarantine, import and export detection, ecological monitoring, prevention and control of invasive species and the like, provides a better solution for quickly identifying the small imported fire ants on site, and has remarkable practical value and popularization and application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of pest detection, and in particular relates to a primer set, method and kit for rapid detection of fire ants (Wasmannia auropunctata) based on LAMP. Background Art

[0002] Traditional polymerase chain reaction (PCR) technology is an important tool in molecular biology, but it has some significant limitations in practical application. For example, PCR requires expensive instrumentation (such as thermal cyclers), long reaction times, and complex operation, which limits its application in field settings or at the grassroots level. Furthermore, PCR technology requires high sample purity and often requires complex nucleic acid extraction and purification steps, further increasing the complexity and cost of detection.

[0003] In recent years, loop-mediated isothermal amplification (LAMP) has gained increasing attention as a novel nucleic acid amplification technology. LAMP utilizes specific primers and a strand-displacing DNA polymerase to efficiently amplify nucleic acids under isothermal conditions (typically 60-65°C). It boasts advantages such as ease of use, high sensitivity, and strong specificity. Currently, LAMP technology has been widely applied in plant disease detection, animal disease detection, food safety testing, and other fields, demonstrating broad application prospects.

[0004] Although LAMP technology has performed well in the field of nucleic acid amplification, its application in field environments still faces some challenges. First, existing LAMP detection systems usually require complex sample pre-processing steps, such as DNA extraction, which not only increases the complexity and time of detection, but also may introduce contamination risks. Secondly, existing technologies have high requirements for the portability of equipment and the ease of operation, which limits its widespread application in field environments. In addition, existing LAMP kits usually do not contain sample release agents, and direct detection without complex DNA extraction cannot be achieved, which further increases the difficulty and cost of field detection. Finally, existing LAMP technology has shortcomings in primer design. Many detection methods require users to design primers themselves, which not only increases the technical threshold, but also may lead to unstable test results. Summary of the Invention

[0005] Traditional PCR technology relies on precision instruments, is complex to operate, and is costly. While existing LAMP technology offers the advantage of isothermal amplification, it still has room for improvement in areas such as sample pretreatment, equipment portability, detection specificity, primer design, and cost control. This invention aims to address the limitations of traditional PCR detection methods in field settings, as well as the shortcomings of existing LAMP technology in detecting small fire ants. It provides a LAMP-based rapid detection primer set, method, and kit for small fire ants. This method and kit are rapid, portable, low-cost, and highly specific, suitable for field use.

[0006] The present invention realizes rapid, portable, and low-cost detection in the field environment through the innovative combination of a commercial isothermal amplification kit, self-designed specific primers, a sample releaser, and a portable constant temperature heater. The kit of the present invention already contains specific primers optimized for the mitochondrial COI gene of the small fire ant, so users do not need to design primers themselves, which lowers the technical threshold and improves the stability and specificity of the detection. In addition, the present invention is equipped with a sample releaser, which can realize rapid crude extraction of DNA in the field, significantly simplifying the sample processing process. By optimizing the kit formula and simplifying the operating process, the present invention also has significant advantages in cost control, providing a more convenient, economical and efficient solution for the detection of small fire ants in the field.

[0007] The LAMP-based rapid detection method and kit for small fire ants of the present invention achieves a breakthrough through the innovative combination of the following core components: 1. Innovative combination of core components: This system integrates a commercial isothermal amplification kit (a product of Hanhai), custom-designed specific primers, a sample release agent (customized by Hanhai), and a portable constant-temperature heater. The commercial isothermal amplification kit provides a stable enzymatic reaction system, while the custom-designed specific primers are optimized for the COI gene sequence of the mitochondrial fire ant, ensuring high detection specificity.

[0008] 2. Simplified sample pre-treatment process: Equipped with a sample release agent, it enables direct lysis and detection without complex DNA extraction, significantly simplifying sample pre-treatment steps, reducing contamination risks, and saving detection time.

[0009] 3. Optimize equipment portability: The portable constant temperature heater provides a precise temperature control environment (65°C), replacing traditional thermal cyclers. The entire equipment weighs less than 1 kg and is suitable for field operations.

[0010] 4. Cost Control: By optimizing the kit formula and simplifying the operating procedures, the cost of a single test is reduced by more than 65% compared to traditional qPCR methods (the traditional method costs 30 yuan per test, while the method of this invention only costs 7 yuan per test).

[0011] The first object of the present invention is to provide a primer set for detecting small fire ants, which includes outer primers F3 and B3, inner primers FIP and BIP, and a loop primer LoopB. The nucleotide sequence of the outer primer F3 is shown in SEQ ID NO.2, The nucleotide sequence of the outer primer B3 is shown in SEQ ID NO.4, The nucleotide sequence of the inner primer FIP is shown in SEQ ID NO.1, The nucleotide sequence of the inner primer BIP is shown in SEQ ID NO.3, The nucleotide sequence of the loop primer LoopB is shown in SEQ ID NO.5.

[0012] The second object of the present invention is to provide a LAMP-based detection kit for small imported fire ants, which includes the primer set and the LAMP nucleic acid amplification reagent.

[0013] Preferably, the LAMP nucleic acid amplification reagent is the RT-LAMP colorimetric premix produced by Wuhan Hanhai Xinzyme Biotechnology Co., Ltd., product number: HMD5204-01.

[0014] The third object of the present invention is to provide a LAMP-based rapid detection kit for small fire ants, which includes the LAMP-based rapid detection kit for small fire ants and a sample releaser.

[0015] Preferably, the sample releaser is a sample releaser produced by Wuhan Hanhai Xinzyme Biotechnology Co., Ltd., product number: HMD3508-1.

[0016] A fourth object of the present invention is to provide a LAMP-based rapid detection method for small fire ants, comprising the following steps: S1. Collect the ant sample to be tested and place it in a centrifuge tube. Grind it, add 800 µL of sample release agent to the centrifuge tube, let it stand for 5 minutes, take 1 µL of the sample solution and add it to the detection reaction centrifuge tube, and react at 65°C for 40 minutes. Each detection reaction centrifuge tube contains 49 µL of LAMP reaction mixture, which consists of 10 µL of Loop-mediated Amplification Buffer with dye, 2.8 µL of RT-Enzymes Mix, 5 µL of primer mixture, and 31.2 µL of nuclease-free water. In the primer mixture system, the concentration of the outer primer F3 is 2 µM, the concentration of the outer primer B3 is 2 µM, the concentration of the inner primer FIP is 16 µM, the concentration of the inner primer BIP is 16 µM, and the concentration of the loop primer LoopB is 4 µM. S2. After the reaction is complete, the color of the reaction solution changes to determine if the sample contains fire ants. If the solution changes from pink to yellow, it is a positive result, indicating that the sample contains fire ants. If the solution does not change color significantly, it is a negative result, indicating that the sample does not contain fire ants.

[0017] Preferably, the Loop-mediated Amplification Buffer with dye and RT-EnzymesMix are components of the RT-LAMP colorimetric premix produced by Wuhan Hanhai New Enzyme Biotechnology Co., Ltd., product number: HMD5204-01.

[0018] Preferably, the sample releaser is a sample releaser produced by Wuhan Hanhai Xinzyme Biotechnology Co., Ltd., product number: HMD3508-1.

[0019] Preferably, the nucleotide sequence of the outer primer F3 is shown as SEQ ID NO.2, the nucleotide sequence of the outer primer B3 is shown as SEQ ID NO.4, the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO.1, the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO.3, and the nucleotide sequence of the loop primer LoopB is shown as SEQ ID NO.5.

[0020] Compared with the prior art, the present invention has the following advantages: 1. Portability: The entire set of equipment is light (less than 1 kg) and easy to carry, making it particularly suitable for field operations without relying on laboratory conditions.

[0021] 2. Universality: No specialized laboratory conditions are required, and the operation is simple, so even non-technical personnel can easily complete the test, expanding the application scope of the technology.

[0022] 3. Economical: It significantly reduces the cost of a single test and improves the economy and accessibility of testing.

[0023] 4. High efficiency: Detection completed within 45 minutes, with a sensitivity of 2.31×10 -3 ng / µL, with a specificity of ≥99%, meeting the needs of rapid identification.

[0024] 5. Innovation: The kit already includes specific primers and DNA sample release agents, eliminating the need for users to design primers or perform complex DNA extraction, reducing technical barriers and operational difficulty.

[0025] The present invention provides a rapid detection method for fire ants in the wild. Through an integrated design, the complex laboratory operation is transformed into a three-step process of "sample → reaction tube → result". The detection is completed within 45 minutes, with a sensitivity of 2.31×10 -3ng / µL, specificity ≥99%; the present invention can be widely used in scenarios such as agricultural quarantine, import and export inspection, ecological monitoring and invasive species prevention and control, providing a better solution for the rapid on-site identification of small fire ants, with significant practical value and promotion and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a comparison chart of the amplification effects of different primer sets based on the NEB chromogenic method; Note: 1-5: the first to fifth primer sets; CK1-CK5: the no-template negative controls corresponding to the first to fifth primer sets.

[0027] Figure 2 This chart compares the qPCR amplification effects of different primer addition amounts and reaction times based on the NEB fluorescence quantitative method; Note: 1-5 represent different primer addition amounts: 3.5 µL, 4.5 µL, 5.5 µL, 2.5 µL, and 1.5 µL, respectively; CK1-CK5: primer addition amounts of 5.5 µL, 4.5 µL, 3.5 µL, 2.5 µL, and 1.5 µL correspond to the corresponding no-template negative controls.

[0028] Figure 3 This is a comparison chart of qPCR sensitivity test based on NEB fluorescence quantitative method; Note: 1 to 7 are different DNA concentrations after dilution: 2.31×10 1 ng / µL, 2.31×10 0 ng / µL, 2.31×10 -1 ng / µL, 2.31×10 -2 ng / µL, 2.31×10 -3 ng / µL, 2.31×10 -4 ng / µL and 2.31×10 -5 ng / µL; CK: no-template negative control.

[0029] Figure 4 This is a comparison chart of qPCR specificity test based on the NEB fluorescence quantitative method; Note: 1 to 5 are the small fire ant, red fire ant, tropical fire ant, double-bone pavement ant and Pharaoh's small house ant; CK: no template negative control.

[0030] Figure 5 This is a comparison chart of the amplification effects of different primer sets based on the Hanhai chromogenic method; Note: 1-5: the first to fifth primer sets; CK1-CK5: the no-template negative controls corresponding to the first to fifth primer sets.

[0031] Figure 6This is an electrophoresis diagram comparing the amplification effects of different primer sets based on the Hanhai chromogenic method; Note: M: 2000 bp marker; 1-5: the first to fifth primer sets; CK1-CK5: the no-template negative controls corresponding to the first to fifth primer sets.

[0032] Figure 7 This is a comparison chart of the amplification effects of different primer addition amounts based on the Hanhai colorimetric method; Note: 1 to 5 represent different primer addition amounts: 3 µL, 4 µL, 5 µL, 6 µL, and 7 µL; CK1 to CK5: No-template negative controls corresponding to different primer addition amounts.

[0033] Figure 8 This is an electrophoresis diagram comparing the amplification effects of different primer addition amounts based on the Hanhai chromogenic method; Note: M: 2000 bp marker; 1-5 are different primer addition amounts: 3 µL, 4 µL, 5 µL, 6 µL, and 7 µL; CK1-CK5: no-template negative controls corresponding to different primer addition amounts.

[0034] Figure 9 This is a comparison chart of the amplification effects of different reaction times based on the Hanhai colorimetric method; Note: 1 to 5 represent different reaction times: 30 min, 35 min, 40 min, 45 min, and 50 min; CK1 to CK5 are template-free negative controls corresponding to different reaction times.

[0035] Figure 10 This is an electrophoretic comparison of the amplification effects at different reaction times based on the Hanhai colorimetric method; Note: M: 2000 bp marker; 1-5 are different reaction times: 30 min, 35 min, 40 min, 45 min and 50 min; CK1-CK5: no-template negative controls corresponding to different reaction times.

[0036] Figure 11 This is a comparison chart of the sensitivity test based on the Hanhai colorimetric method; Note: 1 to 5 are different DNA concentrations after dilution: 2.31×10 1 ng / µL, 2.31×10 0 ng / µL, 2.31×10 -1 ng / µL, 2.31×10 -2 ng / µL and 2.31×10 -3 ng / µL; CK: no-template negative control.

[0037] Figure 12 This is a comparison electrophoresis diagram based on the sensitivity test of the Hanhai colorimetric method; Note: M: 2000 bp marker; 1 to 5 are different DNA concentrations after dilution: 2.31×10 1 ng / µL, 2.31×100 ng / µL, 2.31×10 -1 ng / µL, 2.31×10 -2 ng / µL and 2.31×10 -3 ng / µL; CK: no-template negative control.

[0038] Figure 13 This is a comparison chart of specificity tests based on the Hanhai chromogenic method; Note: 1-5: small fire ant, red fire ant, tropical fire ant, double-bone pavement ant and Pharaoh's small house ant; CK: no template negative control.

[0039] Figure 14 This is a comparative electrophoresis diagram of the specificity test based on the Hanhai chromogenic method; Note: M: 2000 bp marker; 1-5: small fire ant, red fire ant, tropical fire ant, double-bone pavement ant, and Pharaoh's house ant; CK: no-template negative control.

[0040] Figure 15 This is a specificity comparison chart based on the field identification method; Note: 1-3: small fire ants; 4-11: red fire ants, dwarf big-headed ants, Pi's big-headed ants, Hong Kong big-headed ants, common small house ants, Pharaoh's small house ants, black-headed smelly ants and Smith's pavement ants; CK: no template negative control.

[0041] Figure 16 This is the electrophoresis diagram of a sample of the small fire ant based on the field identification method; Note: M: 2000 bp marker; 1-3: small fire ants; CK: no template negative control.

[0042] Figure 17 This is a specificity comparison electrophoresis diagram based on the field identification method; Note: M: 2000 bp marker; 4-11: Red imported fire ant, dwarf big-headed ant, Pi's big-headed ant, Hong Kong big-headed ant, common house ant, Pharaoh's house ant, black-headed smelly ant, and Smith's pavement ant; CK: no-template negative control. DETAILED DESCRIPTION

[0043] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0044] Example 1 1.1 Experimental Materials 1.1.1 Experimental Ants Small fire ants were collected from 8 ant nests in Shantou City, Guangdong Province.

[0045] 1.1.2 Experimental Reagents Tiangen Biochemical Technology (Beijing) Co., Ltd.: Micro-sample genomic DNA extraction kit (DP316).

[0046] Sangon Biotech (Shanghai) Co., Ltd.: Primer synthesis.

[0047] New England Biotech (Beijing) Co., Ltd. kit (hereinafter referred to as the NEB kit): 1. WarmStart Colorimetric LAMP 2X Master Mix with UDG (M1804S). The main components are: Bst 2.0 WarmStart DNA Polymerase, WarmStart RTx Reverse Transcriptase, dUTP, thermosensitive UDG, and a pH indicator. The reaction principle is: During the LAMP reaction, Bst 2.0 WarmStart DNA Polymerase releases a large amount of protons, causing the pH of the reaction system to decrease. The premix contains a visual pH indicator. When the pH value decreases, the reaction solution changes color from pink to yellow, allowing for intuitive identification of positive results. 2. LAMP Fluorescent Dye (B1700S). 3. WarmStart Multi-Purpose LAMP / RT-LAMP 2X Master Mix (with UDG) (M1708S) mainly includes: Bst 2.0 WarmStart DNA Polymerase, WarmStart RTx Reverse Transcriptase, dUTP, thermosensitive UDG, and optimized LAMP buffer. The reaction principle is: the premix and fluorescent dye work synergistically, the target nucleic acid is rapidly amplified during the LAMP reaction, and the fluorescent dye specifically binds to double-stranded DNA and emits a fluorescent signal at a specific wavelength. During the LAMP reaction, as the target nucleic acid is rapidly amplified, the fluorescent signal gradually increases. The real-time fluorescence quantitative PCR instrument (select SYBR / FAM channel) can monitor the changes in fluorescence value in real time, thereby achieving quantitative detection of the LAMP reaction.

[0048] Wuhan Hanhai New Enzyme Biotechnology Co., Ltd. kit (hereinafter referred to as the Hanhai kit): 1. RT-LAMP colorimetric master mix (Cat. No. HMD5204-01). Main components: Loop-mediated Amplification Buffer (with dye), Bst DNA polymerase, and colorimetric dye, and thermostable reverse transcriptase (RT-Enzymes Mix). The reaction principle is that the accumulation of protons produced during the LAMP reaction causes a decrease in pH, which causes the colorimetric dye to change from pink to yellow. 2. Sample release agent (Cat. No. HMD3508-1). The reaction principle is to disrupt cell structure and release intracellular DNA.

[0049] 1.1.3 Main experimental instruments and equipment Grinder, centrifuge, clean bench, electrophoresis instrument, gel imager, PCR instrument, real-time fluorescence quantitative PCR instrument and portable constant temperature heater.

[0050] 1.2 Experimental methods 1.2.1 DNA extraction Ant genomic DNA was extracted using a micro-sample genomic DNA extraction kit (DP316) according to the instructions. During the final elution, 30 μL of sterile deionized water was added to the adsorption membrane as an elution buffer.

[0051] 1.2.2 Primer design and synthesis Based on the sequencing results of the whole mitochondrial genome of the small fire ant, COI PrimerExplorer V5 was used for primer design for the target gene. A total of five primer sets were designed and synthesized in this experiment (Table 1). The first primer set contained outer primers (F3 and B3), inner primers (FIP and BIP), and a loop primer (LoopB), totaling five primers. The other primer sets contained inner primers and outer primers, totaling four primers.

[0052] Table 1 Detailed information of 5 sets of isothermal amplification primers 1.2.3 Experimental reaction system Each primer set was diluted with nuclease-free water, and the primer mixture was prepared at a 10-fold concentration (Table 2). The composition of the NEB chromogenic LAMP reaction system is shown in Table 3 for reactions using the WarmStart Colorimetric LAMP 2X Master Mix with UDG (M1804S). The composition of the NEB fluorescent quantitative LAMP reaction system is shown in Table 4 for reactions using the WarmStart Multi-Purpose LAMP / RT-LAMP 2X Master Mix (with UDG) (M1708S) combined with LAMP Fluorescent Dye (B1700S) for real-time fluorescence quantitative PCR monitoring. The composition of the Hanhai chromogenic LAMP reaction system is shown in Table 5 for reactions using Loop-mediated Amplification Buffer (with dye) (RT-LAMP Chromogenic Master Mix, HMD5204-01).

[0053] Table 2 Composition of primer mixture system Table 3 Composition of NEB colorimetric LAMP reaction system Table 4 Composition of NEB fluorescence quantitative LAMP reaction system Table 5 Composition of Hanhai chromogenic LAMP reaction system 1.3 Optimization of NEB kit isothermal amplification reaction conditions 1.3.1 Primer selection optimization Primer sets were screened using the NEB colorimetric kit. The prepared reaction system was incubated at 65°C in a PCR instrument for 45 minutes. After the reaction, the amplification effect was assessed by observing the color change of the reaction solution. A positive reaction showed a change from pink to yellow, while a negative reaction showed no noticeable color change.

[0054] like Figure 1 As shown in the figure, after preliminary screening, the color change of the first primer group in the NEB colorimetric LAMP reaction was the most obvious, indicating that it had the highest amplification efficiency, while the color changes of the second to fifth primer groups were not obvious, indicating that the amplification efficiency of these primer combinations was low and the pH value could not be significantly changed after the reaction.

[0055] 1.3.2 Optimization of primer addition amount By setting a primer addition gradient from 1.5 to 5.5 μL, collecting fluorescence signals at 30 s intervals, and performing a total of 90 cycles, the results are shown in Figure 2. Figure 2 As shown, when 3.5 µL, 4.5 µL, and 5.5 µL of primer were added, the amplification curve showed a rising trend within 20-25 minutes, indicating a strong amplification signal. When 2.5 µL of primer was added, the amplification curve did not appear until 30-40 minutes, indicating a less pronounced amplification effect than when 3.5 µL was added. Almost no amplification signal was observed within 30 minutes with 1.5 µL. Negative control analysis revealed that significant amplification was observed before 40 minutes with 5.5 µL and 4.5 µL, possibly due to nonspecific amplification. Curves did not rise until after 40 minutes with 2.5 µL and 3.5 µL. Considering both amplification efficiency and nonspecific amplification, 3.5 µL demonstrated high amplification efficiency and low nonspecific amplification, and was selected as the optimal primer addition for subsequent experiments.

[0056] 1.3.3 Response time optimization like Figure 2As shown in the figure, when the primer addition volume is set to 3.5 µL, the amplification reaction reaches peak efficiency, i.e., the plateau phase of the amplification curve, before 35 minutes. However, when the reaction time is extended to 40 minutes or more, an increase in nonspecific amplification is observed, which may interfere with the accuracy of the experimental results. Therefore, in subsequent experimental designs, the reaction time should be controlled within 40 minutes to ensure that the main source of the amplification signal is specific amplification.

[0057] 1.3.4 Sensitivity Exploration The concentration was 2.31×10 2 ng / µL of the small fire ant DNA was diluted 10 times seven times, and the final dilution concentration was 2.31×10 -5 ng / µL, e.g. Figure 3 As shown, at 2.31×10 1 ng / µL, the reaction showed a clear amplification curve between 70 and 80 cycles (about 35 min to 40 min). 0 ng / µL and 2.31×10 -1 ng / µL also showed an amplification curve after 80 cycles, but because nonspecific amplification is likely to occur, only the curve amplified before 80 cycles was considered a valid amplification curve.

[0058] 1.3.5 Specificity exploration The sample concentration range of 200-300 ng / µL was selected from the small fire ant, red fire ant, tropical fire ant, double-bone pavement ant and pharaoh's small house ant for isothermal amplification reaction, such as Figure 4 As shown, only the sample of the small imported fire ant began to show an ascending amplification curve after 50 cycles, reaching an amplification plateau before cycle 70 (35 minutes before the experiment). No obvious amplification curves were observed for the other four ant species (the red imported fire ant, the tropical imported fire ant, the double-bone pavement ant, and the pharaoh's house ant).

[0059] 1.4 Optimization of Isothermal Amplification Reaction Conditions of Hanhai Kit 1.4.1 Primer selection optimization Primer sets were screened using the Hanhai colorimetric kit. The prepared reaction system was incubated at 65°C in a PCR instrument for 40 minutes. After the reaction, the amplification effect was evaluated by observing the color change of the reaction solution. A positive reaction indicated a change from pink to yellow, while a negative reaction showed no noticeable color change.

[0060] After preliminary screening, Hanhai colorimetric method showed that the color change of the first set of primers in the LAMP reaction was the most obvious, such as Figure 5As shown, it has the highest amplification efficiency, while the color changes of the second to fifth primer groups are not obvious, indicating that the amplification efficiency of these primer combinations is low and they cannot cause significant changes in pH after the reaction. The Hanhai colorimetric method further verified the amplification results through gel electrophoresis, as shown in Figure 2. Figure 6 As shown in the figure, the electrophoresis bands of the first primer set were the clearest, while the second to fifth primer sets showed almost no bands. Therefore, the first primer set was selected as the optimal primer combination for subsequent experiments.

[0061] 1.4.2 Optimization of primer addition amount The primer addition amount was set to 3 μL, 4 μL, 5 μL, 6 μL and 7 μL, and the mixture was incubated at 65°C for 40 min. Figure 7 As shown, primer additions between 5 and 7 µL showed a strong color change, while additions of 3 to 4 µL did not produce a significant amplification effect. Negative control analysis revealed color change and band amplification in the 7 µL negative control, likely due to nonspecific amplification. Figure 8 The electrophoresis diagram confirmed the color development results. Considering both amplification efficiency and nonspecific amplification, 5 µL and 6 µL primer additions demonstrated higher amplification efficiency and lower nonspecific amplification. Therefore, 5 µL was selected as the optimal primer addition amount for subsequent experiments.

[0062] 1.4.3 Response time optimization The reaction time gradient was set to 30 min, 35 min, 40 min, 45 min and 50 min, and the incubation was carried out in a 65°C PCR instrument. Figure 9 As shown in the figure, a stronger color change was shown after 40 min, while the color change at 30 min and 35 min was not obvious. In the control group, the negative control at 45 min and 50 min also showed color change. Figure 10 ) may be due to nonspecific amplification caused by a prolonged reaction time, resulting in the appearance of amplified bands. Therefore, 40 min, when color development is more pronounced, was selected as the optimal reaction time for the experiment.

[0063] 1.4.4 Sensitivity Exploration 2.31×10 2 ng / µL of fire ant DNA was diluted 10-fold for five times, and the final dilution concentration was 2.31×10 -3 ng / µL, e.g. Figure 11 As shown in the figure, the five diluted concentrations all changed color, and in the electrophoresis diagram ( Figure 12 ) also verified the presence of the amplified product.

[0064] 1.4.5 Specificity Exploration The specificity of the isothermal amplification reaction was investigated using the Hanhai colorimetric kit. Samples of the fire ant, fire ant, fire ant, double-bone pavement ant, and pharaoh's house ant with a concentration range of 200-300 ng / µL were selected for isothermal amplification reaction. Figure 13 As shown in the figure, only the fire ant sample changed color, while no obvious color change was observed in the other four ant species. Figure 14 ) also verified this result, that is, specific amplification products were only found in the samples of small fire ants, and no amplification products were detected in the samples of other ants.

[0065] 1.5 Field Identification Technology The three methods (colorimetric method, real-time fluorescence quantitative method and electrophoresis) of the two kits all demonstrated the effectiveness of isothermal amplification technology in identifying small fire ants.

[0066] Field identification enables rapid detection of small fire ants, facilitating early detection and implementation of control measures. Based on this, this example combines the Hanhai test kit with primers, sample release agents, and optimized reaction conditions, along with a constant temperature heater and detailed operating instructions, to develop a standalone product.

[0067] 1.5.1 Experimental Materials Ant species with similar body size or body color to the Wasmannia auropunctata invasion area were selected for testing, including the red imported fire ant Solenopsis invicta , Dwarf Ant Pheidole parva , Pi's big-headed ant Pheidole pieli , Hong Kong big-headed ant Pheidole hongkongensis 、Common house ant Monomorium triviale , Pharaoh's House Ant Monomorium pharaonis , black-headed acid ant Tapinoma melanocephalum and Shi's pavement ant Tetramorium smithi All were collected in Guangdong Province.

[0068] 1.5.2 Experimental methods After collecting small fire ant samples in the field, they are placed in clean centrifuge tubes and gently ground using a portable grinding rod (generally 10-30 ants are sampled, depending on the size of the ants). 800 µL of sample release agent is then added to the centrifuge tube. After 5 minutes of simmering, 1 µL of the treated sample is added to the test reaction centrifuge tube. The reaction is incubated at 65°C for 40 minutes. A negative control without template is also included to ensure test accuracy. After the reaction, if the test sample changes color from pink to yellow while the negative control remains unchanged, the sample is confirmed to be a small fire ant.

[0069] 1.5.3 Specificity test like Figure 15 As shown, the color of the fire ant sample changed significantly after the reaction, from pink to yellow. No significant color change was observed in other ant samples using the same detection method. This result shows that the developed product reagent is highly specific to the fire ant and can accurately distinguish the fire ant from other ant species with similar morphology. The amplification results were further verified by gel electrophoresis of the fire ant sample reaction solution. The electrophoresis results showed that only the fire ant sample had a specific amplification product ( Figure 16 ), no amplified products were found for other ant species ( Figure 17 ).

[0070] This study used three methods: real-time fluorescence quantitative PCR, direct chromogenic assay, and electrophoresis verification to evaluate the effectiveness of LAMP technology for identifying small imported fire ants. Experimental results demonstrated that the LAMP method, using primers designed in this invention, is effective for identifying small imported fire ants. Compared to traditional laboratory identification methods, the chromogenic assay is particularly suitable for rapid identification in the field. This method is not only convenient and efficient, but also, as a molecular technique, it does not rely on morphological features for identification, overcoming the limitations of traditional identification methods.

[0071] Isothermal amplification LAMP identification of small fire ants was carried out using two kits from NEB and one kit from Hanhai. By designing five sets of isothermal amplification primers and testing them with two different colorimetric reagents, the first set of designed primers was finally selected as the optimal primer combination for subsequent experiments. In the NEB fluorescence quantitative assay experiment, 3.5 µL of primer addition was determined to be the optimal condition, under which the primers can achieve high amplification efficiency and low non-specific amplification. The Hanhai kit selected 5 µL of primer addition as the optimal condition. In the reaction time optimization experiment, the fluorescence quantitative assay results of the NEB kit showed that the reaction reached a peak amplification efficiency at 40 min. In the sensitivity study, both kits demonstrated efficient detection capabilities, with the minimum detection limit of the NEB fluorescence quantitative assay kit being 2.31×10 -1 ng / µL, the lowest concentration detected by the Hanhai chromogenic assay kit is 2.31×10 -3 ng / µL. In terms of specificity, comparative testing was conducted on the small fire ant, red imported fire ant, tropical imported fire ant, double-bone pavement ant, and Pharaoh's house ant. These ants share some similarities with the small fire ant in terms of body color, biting ability, and body shape. The results showed that only the small fire ant was successfully amplified by both kits, demonstrating the specificity of the LAMP primers and method for detecting the small fire ant.

[0072] A comparison revealed that the Hanhai test kit offers significant advantages in detection efficiency and cost, costing less than 7 yuan per test, approximately one-third the cost of the NEB test kit (market price approximately 30 yuan per test). Therefore, the Hanhai test kit was selected as the basis for further field identification experiments. Combined with a sample releaser and a portable constant-temperature heater, it enables rapid identification of small fire ants in the field without the need for complex equipment or tedious procedures. Furthermore, to verify the specificity and reliability of the detection method, eight ant species, including red imported fire ants and dwarf big-headed ants, were selected for comparison. These ants are common in areas where small fire ants occur or are potentially suitable for their habitat. They are similar in size or color to small fire ants, making identification challenging. The development of this product provides a new tool for rapid on-site detection of small fire ants, facilitating timely monitoring and prevention of their spread.

Claims

1. A primer set for detecting small fire ants, characterized in that: Including outer primers F3 and B3, inner primers FIP and BIP and loop primer LoopB, The nucleotide sequence of the outer primer F3 is shown in SEQ ID NO.2, The nucleotide sequence of the outer primer B3 is shown in SEQ ID NO.4, The nucleotide sequence of the inner primer FIP is shown in SEQ ID NO.1, The nucleotide sequence of the inner primer BIP is shown in SEQ ID NO.3, The nucleotide sequence of the loop primer LoopB is shown in SEQ ID NO.

5.

2. A LAMP-based detection kit for small fire ants, characterized in that: The method comprises the primer set according to claim 1 and a LAMP nucleic acid amplification reagent.

3. The kit according to claim 2, wherein The LAMP nucleic acid amplification reagent is the RT-LAMP colorimetric premix produced by Wuhan Hanhai Xinzyme Biotechnology Co., Ltd., with the product number: HMD5204-01.

4. A rapid detection kit for fire ants based on LAMP, characterized in that: The invention comprises the LAMP-based small fire ant detection kit and a sample releaser as claimed in claim 2.

5. The kit according to claim 4, characterized in that The sample release agent is produced by Wuhan Hanhai Xinzyme Biotechnology Co., Ltd., with the product number: HMD3508-1.

6. A LAMP-based rapid detection method for fire ants, characterized in that: The following steps are involved: S1. Collect the ant sample to be tested and place it in a centrifuge tube. Grind it, add 800 µL of sample release agent to the centrifuge tube, let it stand for 5 minutes, take 1 µL of the sample solution and add it to the detection reaction centrifuge tube, and react at 65°C for 40 minutes. Each detection reaction centrifuge tube contains 49 µL of LAMP reaction mixture, which consists of 10 µL of Loop-mediated Amplification Buffer with dye, 2.8 µL of RT-Enzymes Mix, 5 µL of primer mixture, and 31.2 µL of nuclease-free water. In the primer mixture system, the concentration of the outer primer F3 is 2 µM, the concentration of the outer primer B3 is 2 µM, the concentration of the inner primer FIP is 16 µM, the concentration of the inner primer BIP is 16 µM, and the concentration of the loop primer LoopB is 4 µM. S2. After the reaction is complete, the color of the reaction solution changes to determine if the sample contains fire ants. If the solution changes from pink to yellow, it is a positive result, indicating that the sample contains fire ants. If the solution does not change color significantly, it is a negative result, indicating that the sample does not contain fire ants.

7. The method according to claim 6, wherein The Loop-mediated Amplification Buffer with dye and RT-Enzymes Mix are components of the RT-LAMP colorimetric premix produced by Wuhan Hanhai New Enzyme Biotechnology Co., Ltd., with the product number: HMD5204-01.

8. The method according to claim 6, characterized in that The sample release agent is produced by Wuhan Hanhai Xinzyme Biotechnology Co., Ltd., with the product number: HMD3508-1.

9. The method according to claim 6, wherein The nucleotide sequence of the outer primer F3 is shown as SEQ ID NO.2, the nucleotide sequence of the outer primer B3 is shown as SEQ ID NO.4, the nucleotide sequence of the inner primer FIP is shown as SEQ ID NO.1, the nucleotide sequence of the inner primer BIP is shown as SEQ ID NO.3, and the nucleotide sequence of the loop primer LoopB is shown as SEQ ID NO.5.

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