Diagnostic target and detection primer for wood infection of pine wood nematodes and application of diagnostic target and detection primer

Abstract

The invention discloses a diagnosis target and a detection primer for wood infection of pine wood nematodes and application of the diagnosis target and the detection primer. According to the invention, through data analysis and comparison of miRNA changes with significant differences between a control group and an experimental group at different time points, six miRNAs which only exist in an inoculation group and do not exist in the control group are finally screened out; furthermore, a specific stem-loop DNA probe is designed by taking the six miRNAs as target genes to establish a pine wood nematode detection method, and according to a sensitivity detection result, the novel-miR574 with a nucleotide sequence as shown in SEQ ID No.13 has optimal detection sensitivity when being used as a diagnosis target for wood infection with pine wood nematode diseases; specificity test results show that whether a forest sample is infected with pine wood nematode or not can be specifically detected by using the novel-miR574 as the diagnosis target of the forest infected with the pine wood nematode disease; the invention has an application prospect in prevention and treatment of pine wood nematodes.

Description

TECHNICAL FIELD

[0001] The present application relates to a diagnostic target for forest trees infected with nematodes, in particular to a diagnostic target for forest trees infected with pine wood nematode, detection primers and application thereof, and belongs to the field of molecular detection of pine wood nematode. BACKGROUND

[0002] In the diagnosis or detection of pine trees infected with pine wood nematode, there have been studies using fluid gum method, unmanned aerial vehicle multispectral image analysis, chlorophyll fluorescence analysis and other methods for early diagnosis, but there is still a lack of effective early diagnostic means that can timely block the spread. Traditional detection methods of miRNA such as Northern blotting and qRT-PCR have limitations such as dependence on expensive equipment and complex sample processing, while the newly developed CAL-LAMP method combined with CRISPR-Cas12a and LAMP has high sensitivity (detection limit as low as 1 amol / L) and specificity for specific miRNA, and can detect miRNA in Korean pine 10 days after inoculation (when asymptomatic). However, the application of this field in plant diseases is still relatively small, and early diagnosis is faced with the defects of small size, low abundance and high homology of miRNA.

[0003] In the study of detecting forest trees infected with pine wood nematode using miRNA as a target, 1026 miRNAs were identified by high-throughput sequencing of small RNAs in Korean pine, of which 149 were differentially expressed between the infected group and the control group. These miRNAs are involved in defense pathways, lipid metabolism and other processes. The miRNAs that only exist in the inoculation group were screened, primers were designed and a CAL-LAMP detection system was established, which can specifically detect novel-miR574, novel-miR544 and novel-miR248, and can be detected 10 days after inoculation (asymptomatic). However, this study has some shortcomings, such as the lack of a reference genome in Korean pine, which requires the prediction of new miRNAs from transcriptome data. The functional role of these miRNAs and the interaction mechanism with pine wood nematode have not been verified. The conservation and specificity in other Pinus species are not clear. The induction factors and additional functions of new miRNAs also need further study, and the current research on miRNA of pine trees infected with pine wood nematode is mostly short-term, lacking long-term expression change analysis. SUMMARY

[0004] One of the purposes of the present application is to provide a diagnostic target for forest trees infected with pine wood nematode disease; The second purpose of the present application is to establish a detection method for forest trees infected with pine wood nematode disease using the diagnostic target.

[0005] The third object of the present application provides a kit for diagnosing or detecting whether a forest tree is infected with pine wood nematode.

[0006] To achieve the above object, the main technical scheme adopted by the present application comprises: application of novel-miR574 as a diagnostic target for whether a forest tree is infected with pine wood nematode, wherein the nucleotide sequence of the novel-miR574 is shown in SEQ ID No. 13; wherein the application of the novel-miR574 as a diagnostic target for whether a forest tree is infected with pine wood nematode comprises: (1) designing a specific stem-loop DNA probe for the novel-miR574 as a detection target gene to establish a reverse transcription reaction system to perform reverse transcription reaction of the novel-miR574; (2) adding a SplintR ligase to the reverse transcription reaction product to establish an enzyme ligation reaction system to perform enzyme ligation reaction; (3) adding the ligation product of the enzyme ligation reaction to a LAMP amplification system to perform LAMP amplification reaction; (4) establishing a CRISPR reaction system for the product of the LAMP amplification reaction to perform CRISPR reaction; and determining whether the sample is infected with pine wood nematode according to the fluorescence intensity of the CRISPR reaction product.

[0007] In a preferred embodiment of the present application, the specific stem-loop DNA probe in step (1) is composed of two probes with nucleotide sequences shown in SEQ ID No. 14 and SEQ ID No. 15.

[0008] In a preferred embodiment of the present application, the reverse transcription reaction system in step (1) is: miRNA 1 μL, probe SLP-pam (1 nmol / L) 1 μL, probe SLP (1 nmol / L) 1 μL, 10× SplintR ligase reaction buffer 0.6 μL, RNase-free water 2.4 μL; wherein the nucleotide sequence of the probe SLP-pam is shown in SEQ ID No. 14, and the nucleotide sequence of the probe SLP is shown in SEQ ID No. 15.

[0009] The parameters of the reverse transcription reaction are preferably: 85℃ for 2 minutes, and 37℃ for 5 minutes.

[0010] In a preferred embodiment of the present application, the enzyme ligation reaction system in step (2) is: reverse transcription reaction product 6 μL, SplintR ligase (25 U / μL) 0.1 μL, 10× SplintR ligase reaction buffer 0.4 μL, RNase-free water 3.5 μL; and the reaction conditions of the enzyme ligation reaction are: 37℃ for 15 minutes.

[0011] In a preferred embodiment of the present application, the LAMP amplification system in step (3) is: enzyme-linked product 2 μL, 8 U / μL Bst 2.0 WarmStart DNA polymerase 0.5 μL, 10×ThermoPol reaction buffer 1.0 μL, 20 μmol / L primer FIP 0.4 μL, 20 μmol / L primer BIP 0.4 μL, 2.5 mmol / L dNTPs 1.0 μL, RNase-free water 2.7 μL; wherein the nucleotide sequence of the primer FIP is shown as SEQ ID No. 19, and the nucleotide sequence of the primer BIP is shown as SEQ ID No. 20; and the LAMP amplification reaction conditions are preferably 65 °C for 20 minutes.

[0012] In a preferred embodiment of the present application, the CRISPR reaction system established in step (4) is: LAMP amplification product 10 μL, 10 μM Cas12a (Cpf1) nuclease 0.2 μL, 10×NE Buffer 2.1 2 μL, 10 μmol / L ssDNA reporter 1 μL, 5 μmol / L crRNA 2 μL, 40 U / μL RNase inhibitor 0.4 μL, RNase-free water 4.4 μL; wherein the nucleotide sequence of the ssDNA reporter is: TTATT, a fluorescent group is connected to the 5' end of the reporter, and a fluorescent quenching group is connected to the 3' end of the reporter, the fluorescent group is preferably FAM, and the fluorescent quenching group is preferably BHQ1; and the nucleotide sequence of the crRNA is shown as SEQ ID No. 25.

[0013] In a preferred embodiment of the present application, the reaction conditions of the CRISPR reaction are preferably: 37 °C for 20 minutes, and then 65 °C for 10 minutes.

[0014] In still another aspect of the present application, a kit for detecting whether a forest tree is infected with pine wood nematode disease is provided, which comprises: a reverse transcription system of miRNA; a SplintR ligase reaction system; a LAMP amplification system and a CRISPR reaction system. The reverse transcription system of the miRNA comprises: miRNA, specific stem loop DNA probe SLP-pam, specific stem loop DNA probe SLP, 10x SplintR ligase reaction buffer and RNase-free water; wherein the nucleotide sequence of the miRNA is shown in SEQ ID No. 13, the nucleotide sequence of the specific stem loop DNA probe SLP-pam is shown in SEQ ID No. 14, and the nucleotide sequence of the specific stem loop DNA probe SLP is shown in SEQ ID No. 15.

[0015] The SplintR ligase reaction system comprises: reverse transcription reaction product, SplintR ligase, 10x SplintR ligase reaction buffer and RNase-free water. The LAMP amplification system comprises: enzyme ligation product, Bst 2.0 WarmStart DNA polymerase, 10x ThermoPol reaction buffer, primer FIP, primer BIP, dNTPs and RNase-free water; wherein the nucleotide sequence of the primer FIP is shown in SEQ ID No. 19, and the nucleotide sequence of the primer BIP is shown in SEQ ID No. 20. The CRISPR reaction system comprises: LAMP amplification product, Cas12a (Cpf1) nuclease, 10x NEBuffer, ssDNA reporter gene, crRNA, RNase inhibitor and RNase-free water; wherein the nucleotide sequence of the ssDNA reporter gene is: TTATT, a fluorescent group is connected to the 5' end of the reporter gene, and a fluorescent quenching group is connected to the 3' end of the reporter gene, the fluorescent group is preferably FAM, and the fluorescent quenching group is preferably BHQ1; and the nucleotide sequence of the crRNA is shown in SEQ ID No. 25.

[0016] The tree in the application is preferably a pine tree, and most preferably a Korean pine.

[0017] The application screens 6 miRNAs existing in the inoculation group but not in the control group by data analysis and comparison of miRNA changes of the control group and the experimental group at different time points; the application further establishes a pine wood nematode detection method by using the 6 miRNAs as target genes to design specific stem-loop DNA probes, and according to the sensitivity detection results, it can be seen that the novel-miR574 with the nucleotide sequence as SEQ ID No. 13 has the optimal detection sensitivity as the diagnostic target of the pine wood nematode disease of forest trees; the specific test results show that the novel-miR574 with the nucleotide sequence as SEQ ID No. 13 can specifically detect whether the sample is infected with the pine wood nematode as the diagnostic target of the pine wood nematode disease of forest trees; the detection method established by the application can accurately and sensitively detect whether the forest tree is infected with the pine wood nematode at an early stage, and has important application prospects for early prevention and treatment of the pine wood nematode. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Distribution results of the miRNAs identified in the samples at different time points.

[0019] Figure 2 Another distribution result of the miRNAs identified in the samples at different time points.

[0020] Figure 3 The miRNAs expression down-regulation or up-regulation results of the experimental group at 1st, 5th, 10th and 20th days; A is the miRNAs expression significant down-regulation of the experimental group at 1st, 5th, 10th and 20th days; B is the miRNAs expression significant up-regulation of the experimental group at 1st, 5th, 10th and 20th days.

[0021] Figure 4 Volcano plots of the differentially expressed genes of the experimental group (D01 and D05) and the control group (CK01 and CK05) at 1st and 5th days; A is the Volcano plot of the differentially expressed genes of the experimental group and the control group at 1st day; B is the Volcano plot of the differentially expressed genes of the experimental group and the control group at 5th day; wherein, the red dot represents the up-regulated gene, the blue dot represents the down-regulated gene, and the gray dot represents the non-differentially expressed gene (P<0.05, |logFC|>1.0).

[0022] Figure 5 Volcano plots of the differentially expressed genes of the experimental group (D10 and D20) and the control group (CK10 and CK20) at 10th and 20th days; A is the Volcano plot of the differentially expressed genes of the experimental group and the control group at 10th day; B is the Volcano plot of the differentially expressed genes of the experimental group and the control group at 20th day; the red dot represents the up-regulated gene, the blue dot represents the down-regulated gene, and the gray dot represents the non-differentially expressed gene (P<0.05, |logFC|>1.0).

[0023] Figure 6 Expression level results of selected different miRNAs in different samples; red: present in sample, blue: not present in sample.

[0024] Figure 7 Fluorescence excitation values of different targets (Mean ± SD; n = 3).

[0025] Figure 8 Fluorescence excitation of novel-miR574 at different concentrations.

[0026] Figure 9 Fluorescence excitation of novel-miR544 at different concentrations.

[0027] Figure 10 Fluorescence excitation of novel-miR248 at different concentrations.

[0028] Figure 11 CAS-LAMP specific detection results of novel-miR574.

[0029] Figure 12 Actual sample specific test results of novel-miR574; NTC is red pine without any treatment, CK5 represents red pine on the 5th day after inoculation with water, CK10 represents red pine on the 10th day after inoculation with water; D1 represents red pine on the 1st day after inoculation with Bursaphelenchus xylophilus, D5 represents the 5th day after inoculation with Bursaphelenchus xylophilus, and D10 represents the 10th day after inoculation with Bursaphelenchus xylophilus. DETAILED DESCRIPTION

[0030] The present application will be further described below in conjunction with specific examples, and the advantages and characteristics of the present application will become more apparent as the description proceeds. However, it should be understood that the examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications or replacements all fall within the protection scope of the present application.

[0031] Example 1 Screening of miRNAs as diagnostic targets for pine wood nematode disease in forest trees Through miRNA data analysis (http: / / www.mirbase.org / ), Figure 1 , Figure 2 and Figure 3determined the differentially expressed miRNAs. By longitudinal comparison analysis across multiple time points between the experimental group and the control group, 149 differentially expressed miRNAs were found to meet the strict criteria of |log2(fold change)|≥1, p value <0.05. On the first day after inoculation, 19 miRNAs were significantly up-regulated, and 20 were down-regulated (Fig. 1A). This trend continued to the subsequent time points, with 12 up-regulated and 17 down-regulated miRNAs on the fifth day (Fig. 1B), followed by 15 up-regulated and 12 down-regulated miRNAs on the tenth day (Fig. 1A). Notably, the most significant differential expression occurred on the twentieth day, with 48 up-regulated and 21 down-regulated miRNAs observed (Fig. 1B), indicating the dynamic progression of host-pathogen interactions. Figure 4 A). This trend continued to the subsequent time points, with 12 up-regulated and 17 down-regulated miRNAs on the fifth day (Fig. 1B), followed by 15 up-regulated and 12 down-regulated miRNAs on the tenth day (Fig. 1A). Notably, the most significant differential expression occurred on the twentieth day, with 48 up-regulated and 21 down-regulated miRNAs observed (Fig. 1B), indicating the dynamic progression of host-pathogen interactions. Figure 4 B). Figure 5 A). This trend continued to the subsequent time points, with 12 up-regulated and 17 down-regulated miRNAs on the fifth day (Fig. 1B), followed by 15 up-regulated and 12 down-regulated miRNAs on the tenth day (Fig. 1A). Notably, the most significant differential expression occurred on the twentieth day, with 48 up-regulated and 21 down-regulated miRNAs observed (Fig. 1B), indicating the dynamic progression of host-pathogen interactions. Figure 5 B).

[0032] Based on the expression of these miRNAs in all samples, six miRNAs that were only present in the inoculation group and absent in the control group were selected to design specific primers for sensitivity and specificity tests of the diagnostic target for pine infected by Bursaphelenchus xylophilus. The results of the test found that the six miRNAs that were only present in the inoculation group and absent in the control group were osa-miR1873.2, novel-miR132, novel-miR536, novel-miR544, novel-miR574, and novel-miR248 (Fig. 2). Figure 6

[0033] Test Example 1 Sensitivity test of novel-miR574 as a diagnostic target for pine infected by Bursaphelenchus xylophilus Specific amplification primers and crRNA sequences were designed for the six miRNAs (osa-miR1873.2, novel-miR132, novel-miR536, novel-miR544, novel-miR574, and novel-miR248) selected in Example 1 that were only present in the inoculation group and absent in the control group.

[0034] The designed specific amplification primers and crRNA sequences are shown in Table 1.

[0035] Table 1 Target miRNA sequences, amplification primer sequences, and crRNA sequences

[0036] Note: / P / is a phosphorylation modification.​

[0037] The specific steps of the method are as follows: 1 μL of miRNA, 1 μL of 1 nmol / L SLP, 1 μL of 1 nmol / L SLP-pam, and 0.6 μL of 10× SplintR ligase buffer are added. The final volume of the mixed system is 6 μL, which is composed of 2.4 μL of rnase-free water. 85°C for 2 minutes, 37°C for 5 minutes, 0.1 μL of 25 U / μL SplintR ligase, 0.4 μL of 10× SplintR ligase reaction buffer, and 3.5 μL of rnase-free water are added, and the mixture is incubated at 37°C for 15 minutes to obtain the ligase product; then, 2 μL of ligand is added to the LAMP amplification system, including 0.5 μL of 8 U / μL Bst 2.0 WarmStart DNA polymerase, 1.0 μL of 10× ThermoPol reaction buffer, 0.4 μL of 20 μmol / L FIP. 0.4 μL of 20 μmol / L BIP, 1.0 μL of 2.5 mmol / L dNTPs, 2.7 μL of RNase-free water. The mixture is reacted at 65°C for 20 minutes; finally, 0.2 μL of 10 μM Cas12a nuclease, 2 μL of 10× NE Buffer 2.1, 1 μL of 10 μmol / L ssDNA reporter, 2 μL of 5 μmol / L crRNA, 0.4 μL of 40 μmol / L RNase inhibitor, and 4.4 μL of RNase-free water (10 μL) are added to the CRISPR / Cas12a reaction mixture. Then, the mixture is stored at 37°C for 60 minutes. After the reaction is completed, the fluorescence value is observed.

[0038] The method only requires a pair of stem-loop DNA probes with two key components, including a universal stem-loop structure for standardized amplification and a target-specific single-stranded DNA (ssDNA) sequence complementary to the target miRNA. After hybridization with the target, the ssDNA region of the probe serves as a template for SplintR ligase, which catalyzes the formation of a double stem-loop DNA structure. These ligation products subsequently initiate the LAMP reaction. After the LAMP reaction is completed, 2 µL of the amplification product is added to the CRISPR-Cas12a reaction system, and the fluorescence intensity is observed at 37°C for 60 min.

[0039] The detection results of sensitivity are as follows: Figures 7-10As shown, targets novel-miR574, novel-miR544, and novel-miR248 can all detect the corresponding miRNAs well, and each target can accurately identify its own miRNA. Therefore, novel-miR574, novel-miR544, and novel-miR248 were selected as detection targets for pine wilt disease in forest trees.

[0040] according to Figures 8-10 It is evident that the detection limits for the three detection targets differ. Specifically, when detecting novel-miR574, the fluorescence intensity gradually decreases with increasing concentration dilution, until the concentration is diluted to 10. -8 There is still a clear fluorescence curve at (1 amol / L) Figure 8 The fluorescence intensity of novel-miR544 is 10. -8 The detection at (1 amol / L) was the same as the negative control, producing no fluorescence curve; therefore, the detection limit for Novell-Mir 544 was 10. -7 (10 amol / L) Figure 9 The detection limit of novel-miR248 is low, with a target concentration of 10. -7 No fluorescence is produced at (10 amol / L). The fluorescence value is low at excessively high target concentrations. -3 The fluorescence value was highest at (100 fmol / L). Figure 10 ).

[0041] In summary, the target novel-miR574 exhibits the best detection sensitivity as a detection target.

[0042] Experimental Example 2: Specificity test of novel-miR574 as a diagnostic target for pine wilt disease in forest trees. To investigate the specificity of miRNAs, this study used the novel-miR574 specific stem-loop DNA probe at the same concentration (100 fM) to simultaneously detect novel-miR544-crRNA and novel-miR248-crRNA. These probes were designed to perfectly match their corresponding miRNAs.

[0043] Test results as follows Figure 11As shown, NTC1 uses the novel-miR544 detection system to detect another two target miRNAs (novel-miR574 and novel-miR248). NTC2 is a system for detecting novel-miR574 to detect another two target miRNAs (novel-miR248 and novel-miR544). NTC3 is a system for detecting novel-miR248 to detect another two target miRNAs (novel-miR544 and novel-miR574). Only the target miRNA can connect its corresponding specific stem-loop DNA probe as a splint to initiate subsequent LAMP and produce a clear fluorescent signal. In contrast, the presence of other miRNAs, the specific stem-loop DNA probe cannot be effectively connected, and CRISPR-Cas12a has no target to recognize due to the lack of matching splint template and subsequent LAMP amplification.

[0044] Three target primers (Table 1) were used to detect miRNAs on the needles of inoculated red pine nematodes and control groups, and only novel-miR574 was identified in the samples, which was related to its expression level in the samples. The expression levels of the other two detection targets (i.e., novel-miR544 and novel-miR248) were very low.

[0045] From Figure 12 As can be seen, using the CAL-LAMP method to diagnose red pines without any treatment, red pines inoculated with water, and red pines inoculated with pine nematodes, the sample inoculated with pine nematodes had obvious fluorescence production on the 10th day of inoculation, while the sample without pine nematode inoculation and the red pine without any treatment had no fluorescence production. In summary, novel-miR574 can be used as an early diagnostic target for red pine infection with pine nematode disease.

Claims

1. Use of novel-miR574 as a diagnostic target for whether or not a forest tree is infected with pine wood nematode, wherein, The nucleotide sequence of the novel-miR574 is shown in SEQ ID No.

13.

2. Use according to claim 1, characterized in that, It comprises: (1) designing a specific stem-loop DNA probe for the novel-miR574 as a detection target gene to establish a reverse transcription reaction system for the reverse transcription reaction of the novel-miR574; (2) adding a SplintR ligase to the reverse transcription reaction product to establish an enzyme ligation reaction system for the enzyme ligation reaction; (3) adding the ligation product of the enzyme ligation reaction to a LAMP amplification system for the LAMP amplification reaction; (4) establishing a CRISPR reaction system for the CRISPR reaction using the product of the LAMP amplification reaction; and determining whether the sample is infected with the pine wood nematode according to the fluorescence intensity of the CRISPR reaction product.

3. Use according to claim 2, characterized in that, The specific stem-loop DNA probe in step (1) is composed of two probes with the nucleotide sequences shown in SEQ ID No. 14 and SEQ ID No.

15.

4. Use according to claim 2, characterized in that, The reverse transcription reaction system in step (1) is: miRNA 1 μL, probe SLP-pam 1 μL, probe SLP 1 μL, 10× SplintR ligase reaction buffer 0.6 μL, RNase-free water 2.4 μL; wherein the nucleotide sequence of the probe SLP-pam is shown in SEQ ID No. 14, and the nucleotide sequence of the probe SLP is shown in SEQ ID No.

15.

5. Use according to claim 2, characterized in that, The LAMP amplification system in step (3) is: enzyme ligation product 2 μL, 8 U / μL Bst 2.0 WarmStart DNA polymerase 0.5 μL, 10× ThermoPol reaction buffer 1.0 μL, 20 μmol / L primer FIP 0.4 μL, 20 μmol / L primer BIP 0.4 μL, 2.5 mmol / L dNTPs 1.0 μL, RNase-free water 2.7 μL; wherein the nucleotide sequence of the primer FIP is shown in SEQ ID No. 19, and the nucleotide sequence of the primer BIP is shown in SEQ ID No.

20.

6. Use according to claim 2, characterized in that, The CRISPR reaction system established in step (4) is: LAMP amplification product 10 μL, Cas12a nuclease 0.2 μL, 10× NEBuffer 2.1 2 μL, ssDNA reporter 1 μL, crRNA 2 μL, RNase inhibitor 0.4 μL, RNase-free water 4.4 μL.

7. Use according to claim 6, characterized in that, The nucleotide sequence of the ssDNA reporter is: TTATT, and a fluorescent gene is connected to the 5' end of the reporter, and a fluorescent quenching gene is connected to the 3' end of the reporter; and the nucleotide sequence of the crRNA is shown in SEQ ID No.

25.

8. The use according to claim 1, characterized in that, The forest tree is Pinus koraiensis.

9. A kit for detecting whether a tree is infected with pine wilt disease, characterized by, The kit comprises: a reverse transcription system of miRNA, a SplintR ligase reaction system, a LAMP amplification system and a CRISPR reaction system.

10. The kit of claim 9, wherein The reverse transcription system of miRNA comprises: miRNA, a specific stem-loop DNA probe SLP-pam, a specific stem-loop DNA probe SLP, 10x SplintR ligase reaction buffer and RNase-free water; wherein the nucleotide sequence of the miRNA is shown as SEQ ID No. 13, the nucleotide sequence of the specific stem-loop DNA probe SLP-pam is shown as SEQ ID No. 14, and the nucleotide sequence of the specific stem-loop DNA probe SLP is shown as SEQ ID No.

15. The SplintR ligase reaction system comprises: a reverse transcription reaction product, a SplintR ligase, 10x SplintR ligase reaction buffer and RNase-free water. The LAMP amplification system comprises: an enzyme ligation product, Bst 2.0 WarmStart DNA polymerase, 10x ThermoPol reaction buffer, primer FIP, primer BIP, dNTPs and RNase-free water; wherein the nucleotide sequence of the primer FIP is shown as SEQ ID No. 19, and the nucleotide sequence of the primer BIP is shown as SEQ ID No.

20. The CRISPR reaction system comprises: a LAMP amplification product, Cas12a (Cpf1) nuclease, 10x NEBuffer, ssDNA reporter gene, crRNA, RNase inhibitor and RNase-free water; wherein the nucleotide sequence of the ssDNA reporter gene is: TTATT, a fluorescent gene is connected to the 5' end of the reporter gene, and a fluorescent quencher gene is connected to the 3' end of the reporter gene; and the nucleotide sequence of the crRNA is shown as SEQ ID No.

25. The forest tree is Korean pine.

Images