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

Abstract

The invention discloses a primer probe group and a kit for identifying eutrophic 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 euphorbia hirta comprises an Xc primer probe group; the Xc primer probe group comprises an upstream specific primer with a nucleotide sequence as shown in SEQ ID NO: 1, a downstream specific primer with a nucleotide sequence as shown in SEQ ID NO: 2 and an Xc probe with a nucleotide sequence as shown in SEQ ID NO: 3. And the Xc primer probe group has strong specificity and high sensitivity, and can accurately identify the eutrophic bark beetles.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a primer probe set, a kit and applications thereof for identifying a wood borer. Background Art

[0002] Syntrichoptera: Xyleborus cognatus Blandford belongs to the order Coleoptera, family Curculionidae, subfamily Scolytinae, genus Scolytinae Xyleborus The symbiotic wood beetle is often found in mangroves, but its main damage is not limited to plants in the Rhizophoraceae family. Currently, dozens of host species have been recorded, and its host is very wide. The symbiotic wood beetle's tunnels can reach 10 cm deep into the tree trunk. Large-scale aggregations may cause great damage to the host plants. Some wood beetle species become more harmful after leaving their native habitat, such as the light wood beetle ( Xyleborus glabratus ) did not have a great impact on host plants in its native habitat, but after invading North America, it harmed healthy trees and spread pathogens, causing tree death and resulting in great economic losses.

[0003] In recent years, biodiversity surveys in Guangdong have revealed the damage caused by the common wood beetle. The common wood beetle is a key target for surveys and monitoring. Xyleborus pfeilii , rubber wood beetle Xyleborus affinis , Pseudocyx spp Xyleborus perforans , redwood borer Xyleborus ferrugineus The morphologies of several wood beetle species are very similar, and it is difficult to identify their species based solely on their external morphology, which requires a high level of professional technical knowledge from the identifier.

[0004] Therefore, it is extremely important to develop a method that can efficiently, accurately and sensitively identify wood beetles. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a primer-probe set for identifying the syngenetic wood beetle, which has strong specificity and high sensitivity and can accurately identify the syngenetic wood beetle.

[0006] The invention also provides a kit for identifying the wood borer.

[0007] The present invention also provides applications of the primer probe set or kit.

[0008] The present invention also provides a method for detecting wood borer for purposes other than disease diagnosis and treatment.

[0009] The present invention also provides a system for detecting wood borer borers.

[0010] A primer probe set for identifying a syngenetic wood borer according to the first embodiment of the present invention comprises an Xc primer probe set; The Xc primer probe set includes an upstream specific primer whose nucleotide sequence is shown in SEQ ID NO: 1, a downstream specific primer whose nucleotide sequence is shown in SEQ ID NO: 2, and an Xc probe whose nucleotide sequence is shown in SEQ ID NO: 3.

[0011] The primer probe set according to the embodiment of the present invention has at least the following beneficial effects: The Xc primer probe set of the embodiment can effectively distinguish the syngenetic wood beetle from seven closely related species (granular wood beetle, pine wood beetle, red wood beetle, checkerboard wood beetle, double-toothed wood beetle, rubber wood beetle, and light wood beetle), achieving rapid, accurate and stable detection of the syngenetic wood beetle; it can detect syngenetic wood beetle genomic DNA concentration as low as 1×10 -4 ng / μL of sample.

[0012] The identification of conspecific wood beetles using the Xc primer-probe set overcomes the limitations of traditional morphological testing and is suitable for detecting different stages of conspecific wood beetles. This lays the foundation for molecular classification of the genus and rapid identification of closely related species. This method can be directly applied to the quarantine and identification of conspecific wood beetles during import and export plant quarantine, which is of great significance for protecting agricultural and forestry production safety, maintaining ecological balance, and ensuring public health.

[0013] According to some embodiments of the present invention, the 5' end of the Xc probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0014] According to a second aspect of the present invention, a kit for identifying the syngenetic wood bark beetle comprises the above-mentioned primer probe set.

[0015] According to some embodiments of the present invention, the kit further comprises a real-time fluorescence PCR amplification premix; the real-time fluorescence PCR amplification premix comprises a stabilizer, DNA polymerase, Mg 2+ and at least one of dNTPs. The DNA polymerase is preferably a hot start Taq DNA polymerase.

[0016] According to some embodiments of the present invention, the kit further comprises a universal primer probe set.

[0017] According to some embodiments of the present invention, the universal primer probe set includes an upstream universal primer with a nucleotide sequence as shown in SEQ ID NO: 4, a downstream universal primer with a nucleotide sequence as shown in SEQ ID NO: 5, and a universal probe with a nucleotide sequence as shown in SEQ ID NO: 6.

[0018] The universal primer probe set can amplify the syngenetic wood beetle, grain wood beetle, pine wood beetle, red wood beetle, checkerboard wood beetle, double-toothed wood beetle, rubber wood beetle, and light wood beetle, and has no interference with the Xc primer probe set. It has good amplification efficiency, can effectively improve test efficiency, and reduce reagent and consumable costs.

[0019] According to some embodiments of the present invention, the 5' end of the universal probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0020] According to some embodiments of the present invention, the Xc probe and the universal probe are labeled with different fluorescent reporter groups.

[0021] According to some embodiments of the present invention, the 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.

[0022] According to some embodiments of the present invention, the fluorescence quenching group includes but is not limited to any one of BHQ, TAMRA, MGB and DABCYL.

[0023] According to some embodiments of the present invention, the fluorescent reporter group is HEX, and the fluorescent quencher group is BHQ.

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

[0025] Use of the primer probe set or kit according to the third aspect of the present invention in A1) or A2); A1) Detection or auxiliary detection of wood borer; A2) Prepare products for detecting or assisting in the detection of wood borer.

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

[0027] According to a fourth aspect of the present invention, a method for detecting wood bark borer for purposes other than disease diagnosis and treatment comprises the following steps: Using the DNA of the sample to be tested as a template, fluorescent PCR amplification is performed using the primer probe set described in the first embodiment or the kit described in the second embodiment to obtain an amplification curve; and based on the amplification curve, it is determined whether the sample to be tested contains or is a candidate for containing the wood borer.

[0028] According to some embodiments of the present invention, the amplification program of the fluorescent PCR amplification is: 94°C~96°C 9 min~11 min; 94°C~96°C 13 s~17 s, 60°C 55 s~60 s, collecting fluorescence values, 40 or 45 cycles.

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

[0030] The PCR program used in the present invention is designed based on a two-step real-time fluorescence PCR method, wherein 60°C for 55 s to 60 s (preferably 60°C for 60 s) corresponds to annealing / extension, i.e., annealing and extension are achieved simultaneously.

[0031] According to some embodiments of the present invention, judging whether the sample to be tested contains or is a candidate for containing the congener beetle according to the amplification curve is: if the amplification curve of the Xc primer probe set is an S curve and the Ct value is ≤36, judging that the sample to be tested contains or is a candidate for containing the congener beetle; if the Ct value is >36 or there is no Ct value, judging that the sample to be tested does not contain the congener beetle.

[0032] According to some embodiments of the present invention, in the amplification system of the fluorescent PCR amplification, the concentration of the upstream specific primer is 0.2 μmol / mL~0.5 μmol / mL, preferably 0.3 μmol / mL; the concentration of the downstream specific primer is 0.2 μmol / mL~0.5 μmol / mL, preferably 0.3 μmol / mL; the concentration of the Xc probe is 0.1 μmol / mL~0.3 μmol / mL, preferably 0.2 μmol / mL.

[0033] According to some embodiments of the present invention, in the amplification system of the fluorescent PCR amplification, the concentration of the upstream universal primer is 0.2 μmol / mL~0.5 μmol / mL, preferably 0.3 μmol / mL; the concentration of the downstream universal primer is 0.2 μmol / mL~0.5 μmol / mL, preferably 0.3 μmol / mL; the concentration of the universal probe is 0.1 μmol / mL~0.3 μmol / mL, preferably 0.2 μmol / mL.

[0034] 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, preferably 0.05 U / µL.

[0035] According to some embodiments of the present invention, in the amplification system of the fluorescent PCR amplification, the Mg 2+ The concentration is 3 mmol / mL~5 mmol / mL, preferably 4 mmol / mL.

[0036] 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, preferably 0.2 mmol / mL.

[0037] According to some embodiments of the present invention, in the fluorescent PCR amplification system, the concentration of the DNA of the sample to be tested is 1×10 -5 ng / μL~10 ng / μL, preferably 1×10 -4 ng / μL~10 ng / μL.

[0038] According to a fifth aspect of the present invention, a system for detecting wood borer borers comprises B1) and B2); B1) the primer-probe set described in the embodiment of the first aspect or the kit described in the embodiment of the second aspect; B2) Fluorescence detection instrument.

[0039] According to some embodiments of the present invention, the fluorescence detection instrument may be a fluorescence quantitative PCR instrument.

[0040] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which: Figure 1 The specific detection results of the primer probe set of Example 1 of the present invention; Figure 2 This is the specific detection result of the primer probe set of Comparative Example 1 of the present invention; Figure 3 This is the specific detection result of the primer probe set of Comparative Example 2 of the present invention; Figure 4 is the real-time fluorescence PCR detection result of the universal primer probe set of the present invention; Figure 5 This is the sensitivity test result of the primer probe set of Example 1 of the present invention. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0043] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0044] In the description of the present invention, the terms "comprises" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to the process, method, product or apparatus.

[0045] In this study, all wood beetle samples were morphologically identified and reviewed by Associate Professor Li You of Fujian Agriculture and Forestry University. Basic information on the wood beetle and its related species is shown in Table 1. The main reference books are: (1) Smith SM, Beaver RA, 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. Chinese Economic Insects; Volume 29, Coleoptera: Microptera[M]. Beijing: Science Press, 1984. (3) Lin Wei, Xu Miaofeng, Li You, et al. Description of species of the genus Borrelia in China [J]. Plant Quarantine, 2021.35(2):12-19. (4)Fraedrich S,Harrington T,Rabaglia R,et al. A fungal symbiont ofthe redbay ambrosia beetle causes a lethal wilt in redbay and other Lauraceaein the southeastern United States. Plant Disease,2008,92(2):215-224.; (5)Nugraha M N. Productivity and mortality of Acacia mangium willd.plantation with associated bark beetle and Nitidulid beetle in eastKalimantan, Borneo, Indonesia[D]. University of Missouri--Columbia, 2019.; (6)Ohno S (1990) The Scolytidae and Platypodidae (Coleoptera) fromBorneo found in logs at Nagoya port 1. Research Bulletin of the PlantProtection Service, Japan 26: 83–94.; (7)Browne, F. G. 1961. The biology of Malayan SCOLYTIDAE andPLATPODIDAE. Malyan Forest Records. No. 22. 255 pages. Excerpt is pages 100 –174.; (8) Maiti PK, Saha N (2004) Fauna of India and the Adjacent Countries.Scolytidae: Coleoptera (Bark- and Ambrosia-beetles) (Vol. 1). Part-1.Introduction and Tribe Xyleborini. Zoological Survey of India, Kolkata, 268pp.; (9) Ohno S, Yoneyama K, Nakazawa H, et al. The Scolytidae and Platypodidae (Coleoptera) from Philippines, found in logs at Nayoga port[J]. Research Bulletin of the Plant Protection Service, Japan, 1987 (23): 87-91.; (10) Tiwari KK, Das AK, Roy MKD, et al. On the Wood Borers ofMangroves of Andaman and Nicobar Islands, India, with Note on the GalleryPattern of Some Insect Borers [J]. Records of the Zoological Survey of India,1980, 77: 357-362.

[0046] Table 1: Basic information of the wood beetle and its related species

[0047] In the present invention, the preparation method of the sample DNA template used for identification is as follows: The test specimen (B. truncatula sample) was placed in a 200 μL centrifuge tube, and 60 μL of extraction solution A (buffer containing 1% SDS, 50 mmol / L Tris·HCl, 25 mmol / L NaCl, and 25 mmol / L EDTA) was added. The tube was thoroughly ground with a molten pipette tip, vortexed to mix, and incubated at 65°C for 45 min. After the incubation, an equal volume of extraction solution B (3 mol / L KAc solution, pH 7.2) was added and gently inverted to mix. The tube was placed on ice for 1 h and centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to a new 500 μL centrifuge tube, and 2 volumes of chilled anhydrous ethanol were added. The tube was mixed and frozen at −20°C for 1 h. The tube was then centrifuged at 12,000 rpm for 10 min. The supernatant was discarded, and the precipitate was washed with 400 μL of 75% (v / v) ethanol in water and centrifuged at 12,000 rpm for 10 min. After the alcohol was dried, the precipitate was dissolved in 50 μL of double-distilled water. After measuring the DNA concentration and purity using a microspectrophotometer, the sample DNA template was stored at -20°C for future use.

[0048] Design and screening of primer probe sets The COI sequences of the congeneric wood beetle and its seven closely related species (checkerboard wood beetle, rubber wood beetle, grain wood beetle, red wood beetle, light wood beetle, pine wood beetle, and double-toothed wood beetle) were identified through experimental cloning and were used to study the genetic differences among wood beetle species.

[0049] The COI of the genus Xylophilus varies little between species, at approximately 3% to 7%. Comparison of 658 base-length sequences across species revealed a limited number of sequences suitable for primer and probe design, and the designed primer and probe sequences often exhibited poor specificity, lack of amplification curves, and low sensitivity.

[0050] In this example, a universal primer and probe set (XyT-F / XyT-R / XyT-P) for the wood beetle was designed based on the elongation factor 1a (EF-1a) gene of the wood beetle. Furthermore, a specific primer and probe set (Xc-F571 / Xc-R628 / Xc-P625) for identifying the wood beetle was designed based on the cytochrome oxidase subunit I (COI) gene fragment sequence of the wood beetle. Some failed primer and probe sets (Xc-F280 / Xc-R352 / Xc-P346 and Xc-F64 / Xc-R236 / Xc-P116) were also designed for comparison.

[0051] The nucleotide sequences of each primer-probe set are shown in Table 2.

[0052] Table 2

[0053] Note: In the sequence, S refers to G or C, R refers to G or A, and Y refers to T or C.

[0054] Specificity of primer-probe sets A qPCR reaction system was prepared by mixing the system shown in Table 3 and performing qPCR reactions. Dual real-time fluorescence PCR was also performed using the universal primer probe set and the primer probe set from Example 1 in the same system (the fluorophores for the universal primer probe and the specific primer probe were FAM and HEX, respectively). After qPCR amplification was completed, the test results were determined based on the fluorescence amplification curve. To ensure the reliability of the results, each experiment was repeated at least three times.

[0055] The PCR reaction program was as follows: 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; and storage at 40°C.

[0056] Table 3

[0057] Test results such as Figures 1 to 4 The samples corresponding to each serial number are shown in Table 1.

[0058] The primer probe set of Example 1 has a good amplification curve for the wood borer, with a Ct value of about 14 cycles, and no amplification curve for other closely related species; the primer probe set of Comparative Example 1 has specific amplification for the target samples (1: Xc397-1; 2: Xc397-2), but two non-target species (3: Xpe66 and 8: Xpf419) have amplification tailing at 36 cycles; while the primer probe set of Comparative Example 2 has poor amplification efficiency, no specific amplification curve for the target samples (1: Xc397-1; 2: Xc397-2), and an amplification curve for one non-target species (3: Xpe66) at 34 cycles.

[0059] The results showed that the universal primer-probe set produced typical amplification curves for all 14 samples, demonstrating that this method can effectively extract genomic DNA. Furthermore, there was no interference between the universal primer-probe set and the primer-probe set of Example 1, resulting in good amplification efficiency, effectively improving experimental efficiency and reducing reagent and consumable costs.

[0060] Sensitivity of primer-probe sets Referring to the above-mentioned specific qPCR method, the sensitivity was tested for seven different concentrations of DNA template of the same wood beetle sample Xc397; 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 -4ng / μL, 1×10 -5 ng / μL.

[0061] Test results such as Figure 5 As shown (wherein, 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).

[0062] The template DNA concentration was 1×10 -4 When the amplification curve was 1×10 - 5 ng / μL DNA template, the Ct value of the amplification curve is 34-36 cycles, and the detection signal is weak; the template concentration is 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, indicating that the amplification curve was unstable. This indicates that the detection limit of this method is 1×10 -5 ng / μL, the optimal detection concentration is 1×10 -4 ~10 ng / μL.

[0063] In addition, the sequencing results of the above-mentioned specific real-time fluorescence PCR amplification products were completely consistent with the DNA sequence of the symbiotic wood beetle, further proving the specific amplification of the above-mentioned detection method for the symbiotic wood beetle.

[0064] In summary, the primer-probe set of Example 1 can rapidly, stably, reliably, and highly sensitively identify conspecific beetles. It is suitable for detecting different stages of conspecific beetles and can effectively distinguish conspecific beetles from granular wood beetles, pine wood beetles, red wood beetles, checkerboard wood beetles, double-toothed wood beetles, rubber wood beetles, and light wood beetles. This lays the foundation for the molecular classification of the genus Coleoptera in the family Coleoptera and the rapid identification of closely related species in my country. The research results can be directly applied to the quarantine and identification of conspecific beetles in import and export plant quarantine, which is of great significance for protecting the safety of agricultural and forestry production in my country and maintaining ecological balance.

[0065] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A primer probe set for identifying a wood borer, characterized in that: Includes Xc primer probe set; The Xc primer probe set includes an upstream specific primer having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream specific primer having a nucleotide sequence as shown in SEQ ID NO: 2, and an Xc probe having a nucleotide sequence as shown in SEQ ID NO:

3.

2. The primer probe set according to claim 1, characterized in that: The 5' end of the Xc probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

3. A kit for identifying wood borer beetles, 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 an upstream universal primer whose nucleotide sequence is shown in SEQ ID NO: 4, a downstream universal primer whose nucleotide sequence is shown in SEQ ID NO: 5, and a universal probe whose nucleotide sequence is 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 wood borer; A2) Prepare products for detecting or assisting in the detection of wood borer borers.

7. A method for detecting wood 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; judging whether the sample to be tested contains or is a candidate for containing the wood borer according to 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 9 min~11 min; 94°C~96°C 13 s~17 s, 60°C 55 s~60 s, collecting fluorescence values, 40 or 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 same wood beetle according to the amplification curve is: if the amplification curve of the Xc primer probe group is an S curve and the Ct value is ≤36, it is judged that the sample to be tested contains or is a candidate to contain the same wood beetle; if the Ct value is >36 or there is no Ct value, it is judged that the sample to be tested does not contain the same wood beetle.

10. A system for detecting wood borer borers, characterized in that: The system comprises B1) and B2); B1) The primer-probe set according to claim 1 or 2, or the kit according to any one of claims 3 to 5; B2) Fluorescence detection instrument.

Images