A method for detecting longhinus basilaris based on enzyme-mediated double exponential amplification

Abstract

The application discloses a rapid detection method for longhinipus based on enzyme-mediated double exponential amplification, and designs optimal amplification combinations for specific conserved gene segments of longhinipus. The method can complete nucleic acid double exponential amplification in a constant-temperature single tube without precise temperature-changing equipment. Specificity verification shows that only longhinipus samples produce positive fluorescent signals, and there is no cross amplification with close relatives such as black chest sea weevil. The sensitivity reaches 10 copies / μL, and trace amounts of insect bodies, boring residues and hidden insect state samples in wood can be detected. The whole detection process only takes 30 minutes, and the matched reagent kit can be stably stored at 4 DEG C. The kit has the advantages of high sensitivity, strong specificity, portability and rapidness, and is suitable for port quarantine, field screening of hidden insect pests in forests, and can provide a convenient molecular detection method for longhinipus invasion monitoring and early prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection technology for forest pests, specifically involving a primer and probe combination, a detection kit, and a detection method for rapid detection of the quarantine pest *Bark Borer chinensis* based on enzyme-mediated double exponential amplification (EmDEA) technology. It is suitable for rapid on-site nucleic acid screening of timber and stump samples with concealed insect stages at ports of entry, field monitoring in forest areas, and logging. It can be applied to related technical scenarios such as border biosecurity quarantine, early warning and integrated management of invasive pests in pine forests. Background Technology

[0002] The long-grown bark beetle (Hylurgus ligniperda) is an internationally recognized major forestry quarantine pest, belonging to the subfamily Bark Boraginae of the family Weevilidae in the order Coleoptera. It primarily infests the root collar and base of the trunk of pine trees. Adults and larvae bore into the phloem, forming dense tunnels. They also carry the blue stain fungus, causing blue staining of the xylem and root rot. Mild infestations result in stunted tree growth, while severe infestations can lead to the death of entire pine forests, seriously threatening pine forest ecological security and the forestry economy. This insect exhibits significant generational overlap, and its body, borer debris, and infested timber can spread over long distances across regions. It is extremely difficult to detect, and in the early stages, affected plants often show no obvious symptoms, making it easy to miss or misdiagnose. Once it establishes and spreads, the cost of control is extremely high.

[0003] Currently, the identification methods for bark beetles in long forests are mainly divided into two categories: morphological identification and molecular identification. Traditional morphological identification relies on complete adult specimens, but it is difficult to distinguish between larvae, pupae, and hidden stages within the wood. Furthermore, the morphology of bark beetles is highly similar to that of closely related beetles such as the black-breasted sea beetle and the broad-striped trunk beetle. Identifiers need to have professional taxonomic expertise, and the testing cycle is long, which cannot meet the immediate needs of rapid on-site inspection at ports and large-scale general surveys in forests.

[0004] Current molecular detection technologies for *Bark beetle* mainly rely on conventional PCR, whole-genome sequencing, and SNP population tracing. Conventional PCR requires precise temperature-controlled quantitative PCR instruments, is dependent on the laboratory environment, and has limited portability for field applications. Whole-genome resequencing and SNP marker tracing technologies involve complex sample processing, time-consuming testing, and high equipment investment, making them only suitable for precise laboratory tracing and difficult to achieve rapid batch screening at grassroots levels and ports of entry.

[0005] Isothermal nucleic acid amplification technology, which eliminates the need for thermal cycling equipment, is the mainstream development direction for rapid on-site detection of invasive alien pests. However, there is currently no specific detection system for enzyme-mediated double exponential amplification (EmDEA) of the bark beetle. A complete set of primers, probes, and matching reagent kits that combine high sensitivity, strong species specificity, ease of operation, and rapid on-site detection is lacking, creating technical shortcomings for port quarantine and early detection of hidden pests in forests. Therefore, developing a molecular detection technology for EmDEA of the bark beetle suitable for on-site detection and capable of identifying trace samples at various developmental stages has significant practical application value. Summary of the Invention

[0006] This invention addresses the shortcomings of existing methods for detecting the bark beetle, such as low morphological identification accuracy, reliance on sophisticated temperature-controlled equipment for conventional molecular detection, inability to conduct rapid on-site screening, insufficient sensitivity, and susceptibility to misidentification with closely related beetles. It provides a dedicated primer and probe combination for the bark beetle based on enzyme-mediated double exponential amplification (EmDEA) technology, along with a matching isothermal rapid detection kit, on-site detection methods, and their applications. This invention utilizes specific target sequences to design dedicated primers and probes, achieving double exponential amplification of nucleic acids under isothermal conditions. It eliminates the need for a PCR thermal cycler and combines the advantages of high specificity, high sensitivity, ease of operation, portability, and speed. It is applicable to various challenging quarantine samples, including the beetle body, wood chips, and concealed insect stages in wood, thus overcoming the technical limitations of port quarantine and forest field monitoring.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In one embodiment, the present invention provides a primer-probe combination for detecting Bark Borer based on EmDEA technology, the primer-probe combination comprising DNA upstream primer 1-HL-F1, DNA downstream primer 1-HL-R1, and RNA probe 1-HL-RNA2;

[0009] The nucleotide sequence of 1-HL-F1 is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of 1-HL-R1 is shown in SEQ ID NO.2;

[0011] The 1-HL-RNA2 is a structural sequence, and the nucleotides are shown in SEQ ID NO.3.

[0012] In another embodiment, the 5' end of the RNA probe of the present invention is modified with a fluorescent reporter group FAM, and the 3' end is modified with a fluorescent quencher group BHQ1.

[0013] In one embodiment, the present invention provides a rapid isothermal detection kit for the bark beetle EmDEA, comprising the primer and probe combination described above, and further comprising nucleic acid extraction reagents, isothermal amplification lyophilized enzyme powder, and NTP activation buffer; the lyophilized enzyme powder contains DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase, and signal amplification enzyme.

[0014] In another embodiment, the dry powder accompanying the kit of the present invention can be stably stored for a long time at 4°C, and the total volume of the entire reaction system is 20 μL.

[0015] In one embodiment, the present invention provides a rapid on-site detection method for *Bark beetle*, using the primer-probe combination or the kit for detection, comprising the following steps:

[0016] (1) Extract DNA from the sample to be tested, determine the nucleic acid concentration and convert it to copy number;

[0017] (2) Prepare a 20 μL single-tube EmDEA reaction system by adding upstream and downstream DNA primers, RNA probes, lyophilized enzyme powder, template DNA and activation solution;

[0018] (3) 42℃ isothermal fluorescence amplification, FAM fluorescence signal is collected once every 1 min, and the detection and interpretation are completed in 30 cycles.

[0019] In another embodiment, the reaction system ratio of step (2) of the present invention is as follows: 1 μL of 10 μmol / L upstream primer, 1 μL of 10 μmol / L downstream primer, 1 μL of 1 μmol / L RNA probe, 7 μL of template DNA, and the remainder is made up to 20 μL by lyophilized enzyme powder and 10 μL of activation solution.

[0020] In another embodiment, the sample to be tested in this invention is any type of difficult-to-quarantine sample, such as forest insects, wood chips, or hidden eggs / larvae / pupae inside the wood.

[0021] In another embodiment, the nucleic acid detection gradient setting corresponding to the method of the present invention includes a concentration gradient of 10 copies / μL, and the single reaction system volume is 20μL.

[0022] In another embodiment, the detection specificity of the present invention is manifested in that only the genomic nucleic acid of *Barkhorium longifolium* produces a positive fluorescent amplification signal, and there is no cross-amplification with closely related bark beetles such as *Barkhorium moniliforme*, *Barkhorium floridum*, and *Barkhorium stenoptera*.

[0023] In one embodiment, the present invention provides the application of the primer-probe combination, the kit, or the detection method in port forest quarantine, early screening of hidden pests in pine forests, and monitoring of invasive populations of bark beetles.

[0024] The present invention has the following outstanding beneficial effects:

[0025] (1) Excellent species specificity and no cross-positive interference: The primer and probe combination of the present invention targets the unique conserved gene segment of the long-leaved bark beetle, and only produces a positive fluorescent amplification signal for the nucleic acid of the long-leaved bark beetle. There is no cross-amplification with closely related bark beetles such as the black-breasted sea bark beetle, the flower-shaped bark beetle, and the broad-striped bark beetle. It can accurately distinguish closely related bark beetles with highly similar morphology, avoid the defects of traditional morphological identification that rely on complete adults and are prone to misjudgment, and is suitable for accurate identification of various insect life samples.

[0026] (2) High detection sensitivity, suitable for trace and difficult quarantine samples: The detection system of this invention is set with a nucleic acid concentration gradient of 10 copies / μL, which can detect trace amounts of nucleic acid templates. It can effectively detect difficult samples with extremely low DNA content, such as wood chips, hidden eggs in wood, and larval remains, and solve the problem of insufficient ability of conventional molecular detection to detect trace insect sources.

[0027] (3) Constant temperature reaction, low equipment threshold, suitable for on-site screening: The entire EmDEA amplification process of this invention is kept at a constant temperature of 42℃. No precision temperature-changing equipment such as PCR instruments is required. Fluorescence detection can be completed with just a simple constant temperature device, freeing it from the limitations of laboratory instruments and meeting the portable detection needs of port on-site inspection and large-scale survey in forest areas.

[0028] (4) The kit has strong stability and the system is standardized and easy to promote: The core amplification component of the kit is in the form of lyophilized enzyme powder, which can be stored stably for a long time at 4℃; a standardized 20 μL reaction system is uniformly adopted, and the amount of each component added is fixed in proportion. The operation process is simple and uniform, and no complicated reagent preparation is required. Grassroots quarantine personnel can quickly get started, which is convenient for mass production and grassroots promotion and use.

[0029] (5) The detection process is simple and fast, and the detection covers all insect stages: The entire detection process of this invention can complete signal acquisition and result interpretation in only 30 minutes, which is much faster than traditional PCR and sequencing detection methods. It can simultaneously detect various quarantine samples such as insect bodies, wood chips, hidden eggs, larvae, and pupae in the forest, covering all developmental stages of the bark beetle, and solving the industry pain point of difficulty in identifying hidden insect stages.

[0030] (6) Wide range of applications, supporting biosafety control: The primers, probes, reagent kits and detection methods provided by this invention can be applied simultaneously to port forest quarantine, early screening of hidden pests in pine forests and dynamic monitoring of invasive species. They can provide rapid early warning before large-scale spread of pests, reduce the ecological and economic losses caused by pine forest die-offs, reduce the environmental pressure caused by blind pesticide application, and provide efficient complete molecular detection tools for national border biosafety and forest ecological protection. Attached Figure Description

[0031] Figure 1: Preliminary screening and amplification curves of RNA probes and DNA primers;

[0032] Figure 2: Fluorescence amplification curve of the optimal RNA probe 1-HL-RNA2;

[0033] Figure 3: Fluorescence amplification curve of the optimal DNA upstream primer 1-HL-F1 for screening;

[0034] Figure 4: Fluorescence amplification curves for screening the optimal DNA downstream primer 1-HL-R1;

[0035] Figure 5: Amplification curve for sensitivity verification of the EmDEA detection system of this invention;

[0036] Figure 6: Amplification curve for specificity verification of the EmDEA detection system of the present invention. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified; and the experimental conditions are conventional conditions in the art.

[0038] Example 1: Primer-Probe Combination Screening Experiment

[0039] 1.1 Experimental Materials

[0040] 1) Source of insects: Adult *H. ligniperda* beetles, long-term reared by the Chinese Academy of Quarantine and Inspection Sciences; samples were kindly provided by the Chinese Academy of Quarantine and Inspection Sciences, obtained on November 13, 2025, and brought back to the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences for subsequent experimental testing. Adult samples of *H. ligniperda* from Uruguay, *H. crenatus* from Serbia, *H. varius* from Italy, and *H. glabratus* from Poland were submitted by customs; samples were obtained from the Nanjing Customs Animal and Plant Food Testing Center.

[0041] 2) Reagents: Tiangen tissue genomic DNA extraction kit, ultraRADAR fluorescence isothermal amplification lyophilization kit; lyophilized enzyme powder containing DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase, and signal amplification enzyme; NTP activation buffer;

[0042] 3) Nucleic acid raw materials: Bioinformatics analysis of the whole genome sequence of *Bark Borer chinensis* was performed to screen for a specific gene segment that was highly conserved within the target species and showed no significant homology with other non-target insect sequences. DNA primers HL-2F / R (as shown in Table 1) for conventional PCR detection were designed based on this sequence region. The target fragment was then amplified using ApexHF HS DNA polymerase from Aikerui Biotechnology Co., Ltd. The reaction system and procedure were performed according to the manufacturer's instructions. After obtaining the target band, the amplified product was cloned into the pEASY-Blunt vector and sequenced to obtain the accurate target sequence. Six upstream DNA primers (1-HL-F1~1-HL-F6), six downstream DNA primers (1-HL-R1~1-HL-R6), and six modified RNA probes (1-HL-RNA1~1-HL-RNA6) were used.

[0043] Table 1 Primer sequences suitable for routine PCR detection

[0044]

[0045] Based on the target sequence, DNA primers and RNA probes for EmDEA detection were designed using this sequence region by Suzhou Jingrui Biotechnology Co., Ltd. (Tables 2, 3, and 4). The specific primer sequences are shown in the following tables:

[0046] Table 2. Upstream primer sequences suitable for EmDEA detection

[0047]

[0048] Table 3 shows downstream primer sequences suitable for EmDEA detection.

[0049]

[0050] Table 4. RNA probe sequences suitable for EmDEA detection

[0051]

[0052] Note: The RNA probe is labeled with the reporter group FAM at the 5' end and the quencher group BHQ1 at the 3' end.

[0053] 4) Instrument: BioRad CFX Connect fluorescence quantitative detection system.

[0054] 1.2 Experimental Methods

[0055] DNA was extracted from *Bark beetle* using a tissue genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. The extracted DNA, as measured by NanoDrop, was 126 ng / μL. According to the formula: copy number = 6.02 × 10⁻⁶ 23 ×mass concentration ×10 -9 The calculated nucleic acid copy number of the DNA is 1.15 × 10⁻⁶ (plasmid DNA length × 660). 11 The reaction volume was 20 μL. The experiment was then performed using the UltraRADAR fluorescence isothermal amplification detection blank kit from CrystalRay Technology Co., Ltd. First, 1 μL (10 μmol / L) of upstream DNA primer, 1 μL (10 μmol / L) of downstream DNA primer, and 1 μL (1 μmol / L) of RNA probe (i.e., 3 μL Mix) were added to the UltraRADAR fluorescence isothermal amplification detection blank kit dry powder, which consisted of lyophilized products of DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase, and signal amplification enzyme, along with a lyophilization protectant. The mixture was briefly centrifuged. After centrifugation, 7 μL of template DNA was added along the lower part of the 8-tube strip, and the mixture was briefly centrifuged again and incubated at room temperature for 2 min. After incubation, 10 μL of activation solution consisting of NTP (nucleoside triphosphate) and buffer was added to each 8-tube strip, vortexed for 10 s, and then briefly centrifuged before detection. For quantitative fluorescence detection, the temperature was set at 42℃, with one cycle lasting 1 min, for a total of 30 cycles. Signal acquisition is performed once per cycle, and the reporting group is FAM.

[0056] Screening for optimal combinations of RNA probes and DNA upstream and downstream primers: First, cross-combination tests were performed on DNA primers F4, R4, F5, and R5 used for EmDEA detection to obtain four DNA primer combinations: F4R4 (upstream primer 1-HL-F4, downstream primer 1-HL-R4), F4R5 (upstream primer 1-HL-F4, downstream primer 1-HL-R5), F5R4 (upstream primer 1-HL-F5, downstream primer 1-HL-R4), or F5R5 (upstream primer 1-HL-F4, downstream primer 1-HL-R4). -F5, with the downstream primer being 1-HL-R5), each primer combination contains 6 RNA probes: RNA1 (i.e., RNA probe 1-HL-RNA1), RNA2 (i.e., RNA probe 1-HL-RNA2), RNA3 (i.e., RNA probe 1-HL-RNA3), RNA4 (i.e., RNA probe 1-HL-RNA4), RNA5 (i.e., RNA probe 1-HL-RNA5), or RNA6 (i.e., RNA probe 1-HL-RNA6), for a total of 24 combinations (as shown in Table 5). Detection was performed according to the above reaction system and procedure.

[0057] Table 5 Grouping of DNA primer pairs and RNA probes for preliminary screening

[0058]

[0059] The samples corresponding to the same primer were compared to screen for the RNA probe with the lowest CT value and the strongest endpoint fluorescence signal. Then, based on the amplification results of the four DNA primers matched with this RNA probe, a comprehensive selection was made using both CT value and endpoint fluorescence intensity, prioritizing groups with lower CT values ​​and higher endpoint fluorescence. If a direct difference between two groups was difficult to determine, the group with the lowest CT value was selected. After selecting the optimal RNA probe, the downstream DNA primers and the selected RNA probes were fixed, and six upstream DNA primers were used in rotation to screen for the optimal upstream DNA primer. The reaction system, reaction procedure, and result judgment criteria were the same as above. After selecting the optimal upstream DNA primer, the optimal upstream DNA primers and the optimal RNA probe were fixed, and six downstream DNA primers were used in rotation. The reaction system, reaction procedure, and result judgment criteria were the same as above, ultimately yielding the optimal DNA primer and RNA probe combination.

[0060] The obtained sequences were aligned using DNAMAN software; BioRad CFX Connect was used for alignment. TM The real-time PCR detection system collects data; Sigmaplot 12.5 and AI 2025 are used for plotting.

[0061] 1.3 Experimental Results

[0062] The results showed significant differences in fluorescence amplification signals among different DNA primer combinations. The fluorescence amplification signals of primer combinations 1-HL-F5 and 1-HL-R5 were significantly better than other combinations. Therefore, the fixed DNA primer combinations 1-HL-F5 and 1-HL-R5 were selected for subsequent experiments. Figure 1 The comparison of 24 cross-paired amplification curves shown in the figure reveals that the endpoint fluorescence intensity of the 1-HL-F5+1-HL-R5 primer combination is significantly higher than that of the other groups. Therefore, this DNA primer pair was fixed for subsequent RNA probe screening.

[0063] Six reactions were performed using fixed 1-HL-F5 and 1-HL-R5 probes, each paired with 1-HL-RNA1 to 1-HL-RNA6, with the rest of the system and amplification program remaining unchanged. As shown in Figure 2, 1-HL-RNA2 had a CT value of 3.79, the highest endpoint fluorescence signal, and no amplification signal at NTC, thus confirming 1-HL-RNA2 as the optimal RNA probe.

[0064] With downstream primer 1-HL-R5 and RNA probe 1-HL-RNA2 fixed, upstream primers 1-HL-F1 to 1-HL-F6 were used sequentially for amplification and detection. As shown in Figure 3, the CT value of group 1-HL-F1 was 3.53, indicating the best fluorescence signal, while the negative control showed no signal. Therefore, the optimal upstream primer 1-HL-F1 was selected.

[0065] With upstream primer 1-HL-F1 and RNA probe 1-HL-RNA2 fixed, downstream primers 1-HL-R1 to 1-HL-R6 were used sequentially for amplification. As shown in Figure 4, 1-HL-R1 corresponds to a CT value of 3.58, exhibiting the best fluorescence signal intensity and showing no impurities in the negative result. The optimal primer-probe combination was finally determined to be: 1-HL-F1, 1-HL-R1, and 1-HL-RNA2, with sequences shown in SEQ ID NO. 1-3, respectively.

[0066] Example 2: Preparation of the EmDEA rapid isothermal detection kit for the bark beetle.

[0067] Kit components: Primer and probe combination as described in Example 1; genomic DNA extraction reagent; isothermal amplification lyophilized enzyme powder (containing DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase, signal amplification enzyme, and lyophilization protectant); NTP activation buffer. The lyophilized enzyme powder and primer / probe powder in the kit can be stably stored at 4°C in the dark for extended periods. The standard volume for a single reaction is 20 μL.

[0068] Example 3: Standardized rapid on-site detection method for *Bark beetle*

[0069] 3.1 Experimental Samples

[0070] Quarantine samples including adult insects, wood chips, eggs, larvae, and pupae from inside the wood.

[0071] 3.2 Experimental Procedure

[0072] Step 1: Sample DNA Extraction

[0073] Take the sample to be tested, use the DNA extraction reagent provided with the kit to complete the genomic DNA extraction, determine the nucleic acid concentration using NanoDrop, and calculate the nucleic acid copy number.

[0074] Step 2: Prepare a 20 μL standardized reaction system

[0075] Add the following to the lyophilized enzyme powder in sequence: 1 μL 10 μmol / L 1-HL-F1, 1 μL 10 μmol / L 1-HL-R1, and 1 μL 1 μmol / L 1-HL-RNA2, and centrifuge briefly; add 7 μL template DNA, and centrifuge briefly again; finally, add 10 μL NTP activation buffer, vortex for 10 s, and centrifuge.

[0076] Step 3: Isothermal fluorescence amplification detection

[0077] Place the reaction tube in the fluorescence amplification device, react at a constant temperature of 42 ℃, set 30 cycles, collect the FAM fluorescence signal every 1 min, and interpret the results based on the amplification curve.

[0078] Example 4: Sensitivity Verification Experiment

[0079] 1) Template dilution: The genomic DNA of *Barkhorax longifolius* was serially diluted 10-fold using RNase-free ddH2O to prepare a concentration gradient: 10... 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 copies / μL, 1 copies / μL;

[0080] 2) Group setup: Three biological replicates were set up for each concentration gradient, with pure water NTC serving as a blank control group;

[0081] 3) Amplification and detection: EmDEA amplification was performed according to the system and procedure in Example 3.

[0082] Figure 5 shows the sensitivity gradient amplification curves. Clear positive amplification curves were observed for template concentrations of 10 copies / μL and above; no effective fluorescence signal was observed at 1 copy / μL, and no amplification curve was observed in the blank control. The gradient settings in this experiment included a concentration gradient of 10 copies / μL, and the reaction system was consistently 20 μL.

[0083] Example 5: Specificity Verification Experiment

[0084] 1) Template grouping: DNA from Hylurgus ligniperda (from China and Uruguay), Hylesinus crenatus (from Serbia), Hylesinus varius (from Italy), and Hylurgops glabratus (from Poland) were used as templates, with water as a negative control. EmDEA detection was performed, and pure water was used as a negative control.

[0085] 2) Amplification detection: All templates were detected using the standard system and amplification procedure described in Example 3;

[0086] 3) Figure 6 shows the specific fluorescence amplification curve. In actual sample detection, the DNA of *Bark Borer* samples from both China and Uruguay could be detected by RNA2F1R1 (i.e., the primer-probe combination of RNA probe primer 1-HL-RNA2, upstream primer 1-HL-F1, and downstream primer 1-HL-R1), while other species of bark beetles could not be detected. This indicates that RNA2F1R1 has good specificity and can effectively detect *Bark Borer* from around the world in actual detection without interference from other non-target bark beetles. This proves that this primer-probe combination does not have cross-amplification with closely related beetles and has excellent specificity.

[0087] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A primer-probe combination for detecting the bark beetle based on EmDEA technology, characterized in that, The primer-probe combination includes DNA upstream primer 1-HL-F1, DNA downstream primer 1-HL-R1, and RNA probe 1-HL-RNA2; The nucleotide sequence of the upstream primer 1-HL-F1 of the DNA is shown in SEQ ID NO.1; The nucleotide sequence of the downstream primer 1-HL-R1 is shown in SEQ ID NO.2; The RNA probe 1-HL-RNA2 has a structural sequence, and its nucleotide sequence is shown in SEQ ID NO.

3.

2. The primer-probe combination according to claim 1, characterized in that, The RNA probe is modified with a fluorescent reporter group FAM at its 5' end and a fluorescent quencher group BHQ1 at its 3' end.

3. A rapid isothermal detection kit for the forest bark beetle (EmDEA), characterized in that, The primer-probe combination according to claim 1 or 2 may optionally further include nucleic acid extraction reagent, isothermal amplification lyophilized enzyme powder, and NTP activation buffer; the lyophilized enzyme powder contains DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase, and signal amplification enzyme.

4. The EmDEA isothermal rapid detection kit for the bark beetle according to claim 3, characterized in that, The kit's accompanying dry powder can be stored stably for a long time at 4°C, and the total volume of the entire reaction system is 20 μL.

5. A rapid on-site detection method for *Bark beetle*, characterized in that, The detection using the primer-probe combination of claim 1 or the kit of claim 3 or 4 comprises the following steps: (1) Extract DNA from the sample to be tested, determine the nucleic acid concentration and convert it to copy number; (2) Prepare a 20 μL single-tube EmDEA reaction system by adding upstream and downstream DNA primers, RNA probe, lyophilized enzyme powder, template DNA and activation solution; (3) 42℃ isothermal fluorescence amplification, FAM fluorescence signal is collected once every 1 min, and the detection and interpretation are completed in 30 cycles.

6. The method for rapid on-site detection of *Bark beetle* according to claim 5, characterized in that, The reaction system in step (2) is prepared as follows: 1 μL of 10 μmol / L upstream primer, 1 μL of 10 μmol / L downstream primer, 1 μL of 1 μmol / L RNA probe, 7 μL of template DNA, and the remaining amount is made up to 20 μL by adding lyophilized enzyme powder and 10 μL of activation solution.

7. The method for rapid on-site detection of *Bark Beetle* according to claim 5, characterized in that, The samples to be tested are any type of quarantine samples, including insect bodies, wood chips, and hidden eggs / larvae / pupae inside the wood.

8. The method for rapid on-site detection of *Bark Beetle* according to claim 5, characterized in that, The nucleic acid detection gradient setting corresponding to the method includes a concentration gradient of 10 copies / μL, and the single reaction system volume is 20μL.

9. The method for rapid on-site detection of *Bark beetle* according to claim 5, characterized in that, The detection specificity was shown to be that only the genomic nucleic acid of *Bark beetle* produced a positive fluorescent amplification signal, and there was no cross-amplification with closely related bark beetles such as *Bark beetle*, *Bark beetle*, and *Bark beetle*.

10. The application of the primer-probe combination of claim 1 or 2, the kit of claim 3 or 4, or the detection method of any of claims 5-9 in port forest quarantine, early screening of hidden pests in pine forests, or monitoring of invasive populations of bark beetles.

Images