Primer probe group and kit for identifying red bark beetles and application of primer probe group and kit

Abstract

The invention discloses a primer probe group and a kit for identifying red bark beetles and application of the primer probe group and the kit, and relates to the technical field of molecular biology. The primer probe group for identifying the red material bark beetles comprises a first upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a first downstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a first probe with a nucleotide sequence as shown in SEQ ID NO.3. The primer probe group is strong in specificity and high in sensitivity, overcomes the limitation of traditional morphological detection, and can effectively distinguish red bark beetles from chessboard bark beetles, rubber bark beetles, grain bark beetles, synphytic bark beetles, light bark beetles, pine bark beetles, bidentate bark beetles and other related varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a primer-probe set, a kit and an application for identifying Xyleborus erythrus. Background Art

[0002] Xyleborus erythrus Xyleborus ferrugineus (Fabricius, 1801) belongs to the genus Xyleborus of the subfamily Scolytinae, family Curculionidae, order Coleoptera. It is one of the most widely distributed and economically harmful mycophagous bark beetles in the world. It mainly parasitizes deciduous trees and also parasitizes coniferous trees. Its hosts are very extensive and can damage almost all woody plants. There are more than 180 recorded species so far. The vertical distribution range of this insect is from 0 to 1500 m above sea level. Adult insects usually damage felled trees, injured trees, weak trees or tree stumps with a diameter greater than 10 cm, accelerating or causing the death of trees. While damaging the sapwood, Xyleborus erythrus is also the main disease vector of Ceratocystis fimbriata Ceratocystis fimbriata ), and can cause the occurrence of cocoa wilt disease, resulting in great economic losses. Xyleborus erythrus and Xyleborus intricatus Xyleborus pfeilii ), Xyleborus dispar Xyleborus affinis ), Xyleborus mutilatus Xyleborus perforans ), etc. are extremely similar in morphology. It is difficult to identify their species solely based on external morphology. Therefore, there is an urgent need to develop a convenient and rapid method for identifying Xyleborus erythrus. Summary of the Invention

[0003] The present invention aims to at least solve one of the technical problems existing in the prior art. For this purpose, the present invention provides a primer-probe set for identifying Xyleborus erythrus, which has strong specificity and high sensitivity and can accurately identify Xyleborus erythrus.

[0004] The present invention also provides a kit for identifying Xyleborus erythrus.

[0005] The present invention also provides the application of the above primer-probe set or kit.

[0006] The present invention also provides a method for detecting Xyleborus erythrus for non-disease diagnosis and treatment purposes.

[0007] The present invention also provides a system for detecting Xyleborus erythrus.

[0008] According to an embodiment of the first aspect of the present invention, a primer-probe set for identifying Xyleborus erythrus includes: A first upstream primer having a nucleotide sequence as shown in SEQ ID NO.1, a first downstream primer having a nucleotide sequence as shown in SEQ ID NO.2, and a first probe having a nucleotide sequence as shown in SEQ ID NO.3.

[0009] The primer-probe set according to the embodiments of the present invention has at least the following beneficial effects: The primer-probe set of the embodiment can effectively distinguish *Xyleborus erythroxylon* from 7 related species (*Xyleborus lineatus*, *Xyleborus rubberus*, *Xyleborus mutilatus*, *Xyleborus cohabitans*, *Xyleborus glabratus*, *Xyleborus pini*, *Xyleborus dimidiatus*), realizing rapid, accurate and stable detection of *Xyleborus erythroxylon*; it can detect samples with the genomic DNA concentration of *Xyleborus erythroxylon* as low as 1×10 -5 ng / μL.

[0010] Identifying *Xyleborus erythroxylon* with this primer-probe set can overcome the limitations of traditional morphological detection, be applicable to the detection of different insect states of *Xyleborus erythroxylon*, and lay a foundation for the classification of the genus *Xyleborus* in the family *Curculionidae* of Coleoptera at the molecular level and the rapid identification of related species. It can be directly applied to the quarantine and identification of *Xyleborus erythroxylon* in the quarantine of imported and exported plants, and is of great significance for protecting the safety of agricultural and forestry production, maintaining ecological balance, and ensuring public health safety.

[0011] According to some embodiments of the present invention, the 5' end of the first probe is labeled with a first fluorescent reporter group, and the 3' end is labeled with a first fluorescent quenching group.

[0012] According to some embodiments of the present invention, the first fluorescent reporter group includes but is not limited to any one of FAM, ROX, CY5, HEX, JOE, CY3, NED, TAMRA, TAXAS RED, VIC and TET.

[0013] According to some embodiments of the present invention, the first fluorescent quenching group includes but is not limited to any one of BHQ, TAMRA, MGB and DABCYL.

[0014] According to some embodiments of the present invention, the first fluorescent reporter group is HEX, and the first fluorescent quenching group is BHQ.

[0015] A kit for identifying *Xyleborus erythroxylon* according to the embodiments of the second aspect of the present invention, the kit includes the primer-probe set described in the embodiments of the first aspect of the present invention.

[0016] According to some embodiments of the present invention, the kit further contains a real-time fluorescence PCR amplification premix; the real-time fluorescence PCR amplification premix includes a stabilizer, a DNA polymerase, Mg 2+and at least one of dNTPs. The DNA polymerase is preferably a hot start Taq DNA polymerase.

[0017] According to some embodiments of the present invention, the kit further comprises a universal primer-probe set.

[0018] According to some embodiments of the present invention, the universal primer-probe set includes a second upstream primer with a nucleotide sequence as shown in SEQ ID NO.4, a second downstream primer with a nucleotide sequence as shown in SEQ ID NO.5, and a second probe with a nucleotide sequence as shown in SEQ ID NO.6.

[0019] This universal primer-probe set can amplify Ips acuminatus, Ips granulatus, Ips pini, Ips acuminatus, Ips sexdentatus, Ips bidentatus, Ips heveae, Ips nitidus, and has no interference with the primer-probe set for specifically detecting Ips acuminatus. It has good amplification efficiency, can effectively improve the test efficiency, and reduce the cost of reagents and consumables.

[0020] According to some embodiments of the present invention, the 5' end of the second probe is labeled with a second fluorescent reporter group, and the 3' end is labeled with a second fluorescent quenching group.

[0021] According to some embodiments of the present invention, the first fluorescent reporter group and the second fluorescent reporter group are different.

[0022] According to some embodiments of the present invention, the second fluorescent reporter group includes, but is not limited to, any one of FAM, ROX, CY5, HEX, JOE, CY3, NED, TAMRA, TAXAS RED, VIC, and TET.

[0023] According to some embodiments of the present invention, the second fluorescent quenching group includes, but is not limited to, any one of BHQ, TAMRA, MGB, and DABCYL.

[0024] According to some embodiments of the present invention, the second fluorescent reporter group is FAM, and the second fluorescent quenching group is BHQ.

[0025] Use of the primer-probe set described in the first aspect embodiments of the present invention or the kit described in the second aspect embodiments of the present invention according to the third aspect embodiments of the present invention in A1) or A2); A1) Detecting or assisting in detecting Ips acuminatus; A2) Preparing a product for detecting or assisting in detecting Ips acuminatus.

[0026] According to some embodiments of the present invention, the product is selected from a kit or a chip.

[0027] A method for detecting *Xyleborus compactus* for non-disease diagnosis and treatment purposes according to an embodiment of the fourth aspect of the present invention includes the following steps: Using the DNA of the sample to be tested as a template, performing fluorescence PCR amplification with the primer-probe group described in the embodiment of the first aspect or the kit described in the embodiment of the second aspect above to obtain an amplification curve; judging whether the sample to be tested contains or is a candidate for containing *Xyleborus compactus* according to the amplification curve.

[0028] According to some embodiments of the present invention, the DNA of the sample to be tested is obtained by extraction using the SDS method. Those skilled in the art can also use other well-known methods for extraction as long as the DNA of the sample to be tested can be obtained.

[0029] According to some embodiments of the present invention, the amplification program of the fluorescence PCR amplification is: 94°C - 96°C for 8 min - 10 min; 94°C - 95°C for 5 s - 15 s, 60°C for 55 s - 60 s, collecting fluorescence values, for 40 - 45 cycles.

[0030] According to some embodiments of the present invention, judging whether the sample to be tested contains or is a candidate for containing *Xyleborus compactus* according to the amplification curve includes: if the amplification curve of the primer-probe group is an S curve and the Ct value ≤ 36, determining that the sample to be tested contains or is a candidate for containing *Xyleborus compactus*; if the Ct value of the primer-probe group > 36 or there is no Ct value, determining that the sample to be tested does not contain *Xyleborus compactus*.

[0031] According to some embodiments of the present invention, in the amplification system of the fluorescence PCR amplification, the concentration of the first upstream primer is 0.2 μmol / mL - 0.5 μmol / mL (for example: it can be 0.2 μmol / mL, 0.3 μmol / mL, 0.4 μmol / mL or 0.5 μmol / mL); the concentration of the first downstream primer is 0.2 μmol / mL - 0.5 μmol / mL (for example: it can be 0.2 μmol / mL, 0.3 μmol / mL, 0.4 μmol / mL or 0.5 μmol / mL); the concentration of the first probe is 0.1 μmol / mL - 0.3 μmol / mL (for example: it can be 0.1 μmol / mL, 0.2 μmol / mL or 0.3 μmol / mL).

[0032] According to some embodiments of the present invention, in the amplification system of the fluorescence PCR amplification, The concentration of the second upstream primer is 0.2 μmol / mL to 0.5 μmol / mL (for example: it can be 0.2 μmol / mL, 0.3 μmol / mL, 0.4 μmol / mL or 0.5 μmol / mL); The concentration of the second downstream primer is 0.2 μmol / mL to 0.5 μmol / mL (for example: it can be 0.2 μmol / mL, 0.3 μmol / mL, 0.4 μmol / mL or 0.5 μmol / mL); The concentration of the second probe is 0.1 μmol / mL to 0.3 μmol / mL (for example: it can be 0.1 μmol / mL, 0.2 μmol / mL or 0.3 μmol / mL).

[0033] According to some embodiments of the present invention, in the amplification system of the fluorescent PCR amplification, the concentration of the DNA polymerase is 0.03 U / µL to 0.07 U / µL. For example: it can be 0.03 U / µL, 0.04 U / µL, 0.05 U / µL, 0.06 U / µL or 0.07 U / µL.

[0034] According to some embodiments of the present invention, in the amplification system of the fluorescent PCR amplification, the Mg 2+ concentration is 3 mmol / mL to 5 mmol / mL. For example: it can be 3 mmol / mL, 3.5 mmol / mL, 4 mmol / mL, 4.5 mmol / mL or 5 mmol / mL. The Mg 2+ source includes but is not limited to MgCl2.

[0035] According to some embodiments of the present invention, in the amplification system of the fluorescent PCR amplification, the concentration of the dNTPs is 0.1 mmol / mL to 0.3 mmol / mL. For example: it can be 0.1 mmol / mL, 0.15 mmol / mL, 0.2 mmol / mL, 0.25 mmol / mL or 0.3 mmol / mL.

[0036] According to some embodiments of the present invention, in the amplification system of the fluorescent PCR amplification, the concentration of the DNA of the sample to be tested is higher than 1×10 -5 ng / μL.

[0037] According to some embodiments of the present invention, in the amplification system of the fluorescent PCR amplification, the concentration of the DNA of the sample to be tested is 1×10 -5 ng / μL to 10 ng / μL. For example: it can be 1×10 -5 ng / μL, 1×10-4 ng / μL, 1×10 -3 ng / μL, 1×10 -2 ng / μL, 1×10 -1 ng / μL, 1 ng / μL or 10 ng / μL.

[0038] A system for detecting *Xyleborus glabratus* according to an embodiment of the fifth aspect of the present invention, the system comprising: A detection device, which uses the DNA of a sample to be tested as a template and performs fluorescence PCR amplification using the primer-probe group described in the embodiment of the first aspect above or the kit described in the embodiment of the second aspect above to obtain an amplification curve; An analysis device, which is used to determine whether the sample to be tested contains or is a candidate for containing *Xyleborus glabratus* according to the amplification curve; if the amplification curve of the primer-probe group is an S curve and the Ct value ≤ 36, it is determined that the sample to be tested contains or is a candidate for containing *Xyleborus glabratus*; if the Ct value of the primer-probe group > 36 or there is no Ct value, it is determined that the sample to be tested does not contain *Xyleborus glabratus*.

[0039] According to some embodiments of the present invention, the system further comprises a DNA extraction device for extracting the DNA of the sample to be tested.

[0040] According to some embodiments of the present invention, the fluorescence PCR amplification is performed by a fluorescence detection instrument. The fluorescence detection instrument can be a fluorescence quantitative PCR instrument.

[0041] Other features and advantages of the present invention will be described in the subsequent specification, and, in part, will be obvious from the specification, or will be understood by practicing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The above and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, wherein: Figure 1 Shows the specific detection results of the primer-probe group of Example 1 of the present invention; Figure 2 Shows the specific detection results of the primer-probe group of Comparative Example 1 of the present invention; Figure 3 Shows the real-time fluorescence PCR detection results of the universal primer-probe group of the present invention; Figure 4 Shows the sensitivity detection results of the primer-probe group of Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] The concept of the present invention and the technical effects produced will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0044] For those conditions not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0045] In the description of the present invention, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products, or devices.

[0046] In the description of the present invention, if the first, second, etc. are described, they are only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features or the sequence of the indicated technical features.

[0047] Unless otherwise specified, the nucleotide sequences involved in the present invention are in the order from the 5' end to the 3' end.

[0048] In the present invention, all samples of Xyleborus have been identified by morphology and reviewed by Associate Professor Li You of Fujian Agriculture and Forestry University. The main reference books are as follows: (1)Smith S M, Beaver R A, Cognato A I. A monograph of the Xyleborini (Coleoptera, Curculionidae, Scolytinae) of the Indochinese Peninsula (except Malaysia) and China[J]. ZooKeys, 2020, 983: 1 - 442; (2)Yin Huifen, Huang Fusheng, Li Zhaolin. Economic Insect Fauna of China; Volume 29, Coleoptera, Scolytidae[M]. Beijing: Science Press, 1984; (3)Lin Wei, Xu Miaofeng, Li You, et al. Description of species of the genus Xyleborus in China[J]. Plant Quarantine, 2021, 35(2): 12 - 19; (4)Lin W, Xu M, Gao L, Ruan Y, Lai S, Xu Y, Li Y. New records of two invasive ambrosia beetles (Curculionidae: Scolytinae: Xyleborini) to China. BioInvasions Records, 2021, 10(1): 74–80; (5)Zhou Yijing, Jian Baolei, Sun Jiajia, et al. Prediction of the potential distribution of the quarantine forest pest Xyleborinus saxesenii in China. Plant Quarantine, 2019, 33(1): 65 - 68.

[0049] The basic information of the Xyleborinus samples involved is shown in Table 1.

[0050] Table 1

[0051] In the present invention, the method for preparing the sample DNA template for identification is as follows: Take the test specimen (Xyleborinus sample) and place it in a 200 μL centrifuge tube. Add 60 μL of extraction solution A (a buffer solution containing 1% SDS, 50 mmol / L Tris·HCl, 25 mmol / L NaCl, and 25 mmol / L EDTA), grind it thoroughly with a melted pipette tip, mix well by shaking, and incubate at 65 °C for 45 min; after the incubation, add an equal volume of extraction solution B (a KAc solution with a concentration of 3 mol / L, pH 7.2), slowly invert the tube up and down to mix well, then place it on ice for 1 h, centrifuge at 12000 rpm for 10 min, transfer the supernatant to a new 500 μL centrifuge tube, add 2 volumes of frozen absolute ethanol, mix well, and place it at -20 °C for 1 h; then centrifuge at 12000 rpm for 10 min, discard the supernatant, add 400 μL of 75% (v / v) ethanol aqueous solution to wash the precipitate, centrifuge at 12000 rpm for 10 min, air-dry the alcohol, and dissolve the precipitate with 50 μL of double-distilled water. After measuring the DNA concentration and purity with a micro-spectrophotometer, store the sample (DNA template) at -20 °C for standby.

[0052] Design and screening of the primer-probe group The cytochrome oxidase subunit I (COI) sequences of *Xyleborinus saxesenii* and its seven related species (*Xyleborinus lineatus*, *Xyleborinus rubberus*, *Xyleborinus punctatus*, *Xyleborinus coetus*, *Xyleborinus glabratus*, *Xyleborinus pini*, *Xyleborinus bispinatus*) were identified by experimental cloning and used to study the genetic differences among species of the genus *Xyleborinus*. Unexpectedly, it was found that the COI sequences were highly conserved within species of the genus *Xyleborinus* and showed a certain degree of variation among species, making them good marker genes for studying the phylogenetic relationships among different species of the genus *Xyleborinus*.

[0053] In view of this, a set of universal primer-probes for *Xyleborinus* (XyT-F / XyT-R / XyT-P) was designed based on the elongation factor 1a (EF-1a) of *Xyleborinus*, and a set of specific primer-probes for identifying *Xyleborinus saxesenii* (Xf-F61 / Xf-R334 / Xf-P328) was designed based on the COI gene fragment sequence of *Xyleborinus saxesenii*. Moreover, a set of primer-probes with design failure (Xf-F92 / Xf-R376 / Xf-P328) was cited for comparison.

[0054] The nucleotide sequences of each set of primer-probes are shown in Table 2.

[0055] Table 2

[0056] Note: In the sequences, S refers to G or C, R refers to G or A, Y refers to T or C, and K refers to G or T.

[0057] Specificity of the primer-probe sets The qPCR reaction system was obtained by mixing according to the system shown in Table 3, and the qPCR reaction was carried out. The universal primer-probe set and the primer-probe set of Example 1 were subjected to duplex real-time fluorescence PCR (the fluorescent groups of the universal primer-probe and the specific primer-probe are FAM and HEX respectively). After the qPCR amplification was completed, the test results were determined according to the fluorescence amplification curve. To ensure the reliability of the results, each experiment was repeated more than three times.

[0058] The PCR reaction program was: denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s, annealing / extension at 60 °C for 60 s, collecting fluorescence values, 40 cycles; preservation at 40 °C.

[0059] Table 3

[0060] The detection results are as Figures 1 - 2 shown. The corresponding samples for each serial number are shown in Table 1.

[0061] The primer-probe set of Example 1 had good amplification curves for *Xyleborus compactus* (1: Xf337; 2: XfLL), with Ct values around 15 cycles, and no amplification curves for other related species; while the primer-probe set of Comparative Example 1 had no specific amplification for *Xyleborus compactus* and other related species. This indicates that the primer-probe set of Example 1 has good specificity.

[0062] The results showed that the universal primer-probe set had typical amplification curves for all 14 samples, but no specific amplification for the control group with water as the reference. This also indicates that the experimental method can effectively extract the genomic DNA of *Xyleborus*. Moreover, in the dual real-time fluorescence PCR, there was no interference between the universal primer-probe set and the primer-probe set of Example 1, with good amplification efficiency, effectively improving the test efficiency and reducing the cost of reagents and consumables.

[0063] Sensitivity of the primer-probe set Referring to the qPCR method described in "Specificity of the primer-probe set" above, the sensitivity of the primer-probe set of Example 1 was detected using 7 different concentrations of DNA templates of *Xyleborus compactus* sample XfLL; among them, the DNA template concentrations were 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 1×10 -2 ng / μL, 1×10 -3 ng / μL, 1×10 -4 ng / μL, 1×10 -5 ng / μL.

[0064] The detection results are as Figure 4 shown (where 1: 10 ng / μL; 2: 1 ng / μL; 3: 0.1 ng / μL; 4: 1×10 -2 ng / μL; 5: 1×10 -3 ng / μL; 6: 1×10 -4 ng / μL; 7: 1×10 -5 ng / μL).

[0065] When the template DNA concentration was 1×10 -4 ~10 ng / μL, good amplification curves could be amplified; for the DNA template of 1×10 - 5 ng / μL, the Ct value of the amplification curve was between 34 - 36 cycles, and the detection signal was weak; when the template concentration was lower than 1×10 -5 ng / μL, there was no amplification curve or the Ct value of the amplification curve was greater than 36 cycles due to insufficient sample DNA content, and there were unstable amplification curve situations. This indicates that the detection limit of this method is 1×10 -5 ng / μL, and the optimal detection concentration is 1×10 -4~10 ng / μL.

[0066] In addition, the sequencing results of the above-mentioned specific real-time fluorescence PCR amplification products are completely consistent with the DNA sequence of *Xyleborus saxesenii*, further proving the specific amplification of the above detection method for *Xyleborus saxesenii*.

[0067] In summary, the primer-probe set of Example 1 can quickly, stably and reliably identify *Xyleborus saxesenii* with high sensitivity, is applicable to the detection of different insect states of *Xyleborus saxesenii*, and can effectively distinguish *Xyleborus saxesenii* from *Xyleborus mutilatus*, *Xyleborus pini*, *Xyleborus rubidus*, *Xyleborus tabulaeformis*, *Xyleborus bispinatus*, *Xyleborus rubber* and *Xyleborus glabratus*. It lays a foundation for the classification of the genus *Xyleborus* in the family *Scolytidae* of Coleoptera at the molecular level and the rapid identification of related species. The research results can be directly applied to the quarantine and identification of *Xyleborus saxesenii* in the quarantine of imported and exported plants, which is of great significance for protecting the safety of agricultural and forestry production and maintaining ecological balance.

[0068] The above has described the embodiments of the present invention in detail in conjunction with the embodiments, but the present invention is not limited to the above embodiments. Various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the art.

Claims

1. A primer probe set for identifying redwood borer, characterized in that: include: The nucleotide sequence of the first upstream primer is shown as SEQ ID NO.1, the nucleotide sequence of the first downstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the first probe is shown as SEQ ID NO.

3.

2. The primer probe set according to claim 1, characterized in that: The 5' end of the first probe is labeled with a first fluorescent reporter group, and the 3' end is labeled with a first fluorescent quencher group; Preferably, the first fluorescent reporter group is selected from FAM, ROX, CY5, HEX, JOE, CY3, NED, TAMRA, TAXASRED, VIC or TET; Preferably, the first fluorescence quenching group is selected from BHQ, TAMRA, MGB or DABCYL.

3. A kit for identifying redwood borer, characterized in that: The kit comprises the primer probe set according to claim 1 or 2.

4. The kit according to claim 3, characterized in that The kit also includes a universal primer probe set; The universal primer probe set includes a second upstream primer having a nucleotide sequence as shown in SEQ ID NO.4, a second downstream primer having a nucleotide sequence as shown in SEQ ID NO.5, and a second probe having a nucleotide sequence as shown in SEQ ID NO.

6.

5. The kit according to claim 3, characterized in that The kit also contains a real-time fluorescent PCR amplification premix; the real-time fluorescent PCR amplification premix includes a stabilizer, a DNA polymerase, Mg 2+ and at least one of dNTPs.

6. Use of the primer probe set according to claim 1 or 2, or the kit according to any one of claims 3 to 5 in A1) or A2); A1) Detection or auxiliary detection of redwood borer; A2) Preparation of products for detecting or assisting in the detection of redwood borer.

7. A method for detecting redwood borer for purposes other than disease diagnosis and treatment, characterized in that: The following steps are involved: Using the DNA of the sample to be tested as a template, fluorescent PCR amplification is performed using the primer probe set described in claim 1 or 2, or the kit described in any one of claims 3 to 5 to obtain an amplification curve; and judging whether the sample to be tested contains or is a candidate for containing the redwood borer based on the amplification curve.

8. The method according to claim 7, characterized in that The amplification program of the fluorescent PCR amplification is: 94°C~96°C for 8 min~10 min; 94°C~95°C for 5 s~15 s, 60°C for 55 s~60 s, collecting fluorescence values, and 40~45 cycles.

9. The method according to claim 7, characterized in that: Judging whether the sample to be tested contains or is a candidate to contain the redwood beetle according to the amplification curve includes: if the amplification curve of the primer probe group is an S curve and the Ct value is ≤36, judging that the sample to be tested contains or is a candidate to contain the redwood beetle; if the Ct value of the primer probe group is >36 or there is no Ct value, judging that the sample to be tested does not contain the redwood beetle.

10. A system for detecting redwood borer, characterized in that: The system comprises: A detection device for performing fluorescent PCR amplification using the DNA of a sample to be tested as a template and the primer probe set according to claim 1 or 2, or the kit according to any one of claims 3 to 5, to obtain an amplification curve; An analysis device is used to determine whether the sample to be tested contains or is a candidate for containing the redwood beetle according to the amplification curve; if the amplification curve of the primer probe group is an S curve and the Ct value is ≤36, it is determined that the sample to be tested contains or is a candidate for containing the redwood beetle; if the Ct value of the primer probe group is >36 or there is no Ct value, it is determined that the sample to be tested does not contain the redwood beetle.

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