Diagnostic target for pine wood nematode infection of forest trees, detection primer and application thereof

Abstract

The application discloses a diagnosis target of pine wood nematode infection of forest trees, a detection primer and application thereof. Through data analysis and comparison of miRNA changes with significant differences of the control group and the experimental group at different time points, six miRNAs existing only in the inoculation group but not in the control group are finally screened; further, specific stem loop DNA probes are designed by taking the six miRNAs as target genes to establish a detection method of the pine wood nematode, and according to the sensitivity detection result, it can be seen that the novel-miR574 with the nucleotide sequence shown in SEQ ID No. 13 as the diagnosis target of the pine wood nematode infection of the forest trees has the optimal detection sensitivity; the specificity test result shows that the novel-miR574 as the diagnosis target of the pine wood nematode infection of the forest trees can specifically detect whether the forest tree sample is infected with the pine wood nematode; and the application has application prospect for the prevention and treatment of the pine wood nematode.

Description

Technical Field

[0001] This invention relates to diagnostic targets for nematode infection in forest trees, and more particularly to diagnostic targets, detection primers and their applications for pine wood nematode infection in forest trees, belonging to the field of molecular detection of pine wood nematode. Background Technology

[0002] Regarding the diagnosis or detection of pine wilt disease in pine trees, existing studies have employed methods such as fluid resin assays, UAV multispectral image analysis, and chlorophyll fluorescence analysis for early diagnosis. However, effective early diagnostic methods that can promptly block transmission are still lacking. Traditional methods for detecting miRNAs, such as Northern blotting and qRT-PCR, have limitations such as reliance on expensive equipment and complex sample processing. The newly developed CRISPR-Cas12a combined with LAMP method, CAL-LAMP, exhibits high sensitivity (detection limit as low as 1 amol / L) and specificity for specific miRNAs, and can detect miRNAs in Korean pine as early as 10 days after inoculation (when asymptomatic). However, its application in plant diseases is still limited, and early diagnosis faces challenges such as small miRNA size, low abundance, and high homology.

[0003] In a study using miRNAs to detect pine wood nematode infection in forest trees, high-throughput sequencing of small RNAs in Korean pine identified 1026 miRNAs, of which 149 were differentially expressed between the infected and control groups. These miRNAs are involved in defense pathways and lipid metabolism. MiRNAs present only in the inoculated group were screened, primers were designed, and a CAL-LAMP detection system was established, specifically detecting novel-miR574, novel-miR544, and novel-miR248, detectable as early as 10 days post-inoculation (asymptomatic). However, this study has limitations. For example, Korean pine lacks a reference genome, requiring transcriptome data to predict new miRNAs; the functional roles of these miRNAs and their interaction mechanisms with pine wood nematodes have not been verified; their conservation and specificity in other pine species are unclear; further research is needed on the inducing factors and additional functions of new miRNAs; and current studies on miRNAs in pine trees infected with pine wood nematodes are mostly short-term, lacking long-term expression change analysis. Summary of the Invention

[0004] One objective of this invention is to provide a diagnostic target for pine wilt disease in forest trees; A second objective of this invention is to establish a detection method for pine wilt disease in forest trees using the aforementioned diagnostic target.

[0005] A third objective of this invention is to provide a kit for diagnosing or detecting whether trees are infected with pine wood nematode.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include: The application of novel-miR574 as a diagnostic target for whether forest trees are infected with pine wilt disease includes: (1) designing a specific stem loop DNA probe with novel-miR574 as the detection target gene to establish a reverse transcription reaction system for novel-miR574 reverse transcription reaction; (2) adding SplintR ligase to the reverse transcription reaction product to establish an enzyme ligation reaction system for enzyme ligation reaction; (3) adding the ligation product of the enzyme ligation reaction to the LAMP amplification system for LAMP amplification reaction; (4) establishing a CRISPR reaction system with the product of the LAMP amplification reaction for CRISPR reaction; and determining whether the sample is infected with pine wilt disease based on the fluorescence intensity of the CRISPR reaction product.

[0007] In a preferred embodiment of the present invention, the specific stem-loop DNA probe in step (1) consists 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 invention, the reverse transcription reaction system in step (1) is as follows: 1 μL miRNA, 1 μL probe SLP-pam (1 nmol / L), 1 μL probe SLP (1 nmol / L), 0.6 μL 10× SplintR ligase reaction buffer, and 2.4 μL RNase-free water; 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 preferred parameters for the reverse transcription reaction are: 85°C for 2 minutes and 37°C for 5 minutes.

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

[0011] In a preferred embodiment of the present invention, the LAMP amplification system in step (3) is as follows: 2 μL of enzyme ligation product, 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 primer FIP, 0.4 μL of 20 μmol / L primer BIP, 1.0 μL of 2.5 mmol / L dNTPs, and 2.7 μL of RNase-free water; wherein the nucleotide sequence of primer FIP is shown in SEQ ID No. 19, and the nucleotide sequence of primer BIP is shown in SEQ ID No. 20; the preferred conditions for the LAMP amplification reaction are 65 °C for 20 minutes.

[0012] In a preferred embodiment of the present invention, the CRISPR reaction system established in step (4) is as follows: 10 μL of LAMP amplification product, 0.2 μL of 10 μM Cas12a (Cpf1) nuclease, 2.12 μL of 10× NE Buffer, 1 μL of 10 μmol / L ssDNA reporter gene, 2 μL of 5 μmol / L crRNA, 0.4 μL of 40 U / μL RNase inhibitor, and 4.4 μL of RNase-free water; wherein, the nucleotide sequence of the ssDNA reporter gene is TTATT, a fluorescent gene is attached to the 5' end of the reporter gene, and a fluorescence quenching gene is attached to the 3' end of the reporter gene, the fluorescent group is preferably FAM, and the fluorescence quenching gene is preferably BHQ1; the nucleotide sequence of the crRNA is shown in SEQ ID No. 25.

[0013] In a preferred embodiment of the present invention, the reaction conditions for the CRISPR reaction are preferably: reaction at 37 °C for 20 minutes, followed by storage at 65 °C for 10 minutes.

[0014] Another aspect of the present invention provides a kit for detecting whether trees are infected with pine wilt disease, the kit comprising: a miRNA reverse transcription system; a SplintR ligase reaction system; a LAMP amplification system and a CRISPR reaction system; The miRNA reverse transcription system comprises: miRNA, a specific stem-loop DNA probe SLP-pam, a specific stem-loop DNA probe SLP, 10× 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 includes: reverse transcription reaction product, SplintR ligase, 10×SplintR ligase reaction buffer, and RNase-free water; The LAMP amplification system comprises: enzyme ligation product, Bst 2.0 WarmStart DNA polymerase, 10×ThermoPol reaction buffer, primer FIP, primer BIP, dNTPs, and RNase-free water; wherein the nucleotide sequence of primer FIP is shown in SEQ ID No. 19, and the nucleotide sequence of primer BIP is shown in SEQ ID No. 20; The CRISPR reaction system comprises: LAMP amplification product, Cas12a (Cpf1) nuclease, 10×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 attached to the 5' end of the reporter gene, and a fluorescence quenching gene is attached to the 3' end of the reporter gene, wherein the fluorescent group is preferably FAM, and the fluorescence quenching gene is preferably BHQ1; the nucleotide sequence of the crRNA is shown in SEQ ID No. 25.

[0016] The trees mentioned in this invention are preferably pine trees, and most preferably red pine.

[0017] This invention, through data analysis and comparison of miRNA changes with significant differences between the control and experimental groups at different time points, ultimately screened out six miRNAs that were present only in the inoculation group and absent in the control group. Furthermore, this invention uses these six miRNAs as target genes to design specific stem-loop DNA probes to establish a detection method for pine wilt disease. Sensitivity test results show that novel-miR574 (SEQ ID No. 13) has the optimal detection sensitivity as a diagnostic target for pine wilt disease in forest trees. Specificity test results indicate that novel-miR574 (SEQ ID No. 13) can specifically detect whether a sample is infected with pine wilt disease. The detection method established in this invention can accurately and sensitively detect whether forest trees are infected with pine wilt disease in the early stages, and has significant application prospects for the early control of pine wilt disease. Attached Figure Description

[0018] Figure 1 The distribution of miRNAs identified in samples at different time points.

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

[0020] Figure 3 A represents the downregulation or upregulation of miRNA expression in the experimental group on days 1, 5, 10, and 20; B represents the significant downregulation of miRNA expression in the experimental group on days 1, 5, 10, and 20.

[0021] Figure 4 Volcano plots of differentially expressed genes in the experimental groups (D01 and D05) and control groups (CK01 and CK05) on days 1 and 5; A is the Volcano plot of differentially expressed genes in the experimental group and control group on day 1; B is the Volcano plot of differentially expressed genes in the experimental group and control group on day 5; where red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent genes with no differential expression (P<0.05, |logFC|>1.0).

[0022] Figure 5 Volcano plots of differentially expressed genes in the experimental group (D10 and D20) and control group (CK10 and CK20) at days 10 and 20; A is the Volcano plot of differentially expressed genes in the experimental group and control group at day 10; B is the Volcano plot of differentially expressed genes in the experimental group and control group at day 20; red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent genes with no differential expression (P<0.05, |logFC|>1.0).

[0023] Figure 6 The results show the expression levels of different selected miRNAs in different samples; red: present in the sample, blue: not present in the sample.

[0024] Figure 7 The fluorescence excitation values ​​for different target sites are (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 The results are for CAS-LAMP specific detection of novel-miR574.

[0029] Figure 12 The results are the actual sample specificity test results for novel-miR574; NTC is untreated Korean pine, CK5 represents Korean pine on day 5 after inoculation with water, CK10 represents Korean pine on day 10 after inoculation with water; D1 represents Korean pine on day 1 after inoculation with pine wilt nematodes, D5 represents nematodes on day 5 after inoculation with pine wilt nematodes, and D10 represents Korean pine on day 10 after inoculation with pine wilt nematodes. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0031] Example 1: Screening of miRNAs as diagnostic targets for pine wilt disease in forest trees Analysis of miRNA data ( Figure 1 , Figure 2 and Figure 3The differentially expressed miRNAs were identified. Through longitudinal comparisons between the experimental and control groups across multiple time points, 149 differentially expressed miRNAs were found to meet the stringent criteria of |log2(fold change)|≥1 and p-value <0.05. On day 1 post-inoculation, 19 miRNAs were significantly upregulated, and 20 were downregulated (…). Figure 4 A). This trend continued to the subsequent time point, with 12 miRNAs upregulated and 17 downregulated on day 5. Figure 4 B), followed by 15 upregulated and 12 downregulated miRNAs on day 10. Figure 5 A). Notably, the most significant differential expression occurred on day 20, with 48 upregulated miRNAs and 21 downregulated corresponding miRNAs observed. Figure 5 B), which indicates the dynamic progression of host-pathogen interactions.

[0032] Based on the expression of these miRNAs in all samples, six miRNAs present only in the inoculation group and absent in the control group were selected to design specific primers for detection, and sensitivity and specificity tests were conducted on the diagnostic targets of pine wilt disease in forest trees. The results showed that the six miRNAs present only 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. Figure 6 ).

[0033] Example 1: Sensitivity test of novel-miR574 as a diagnostic target for pine wilt disease in forest trees. 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 present only in the inoculation group and not in the control group.

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

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

[0036] Note: / P / indicates phosphorylation modification.

[0037] The specific steps of this method are as follows: Add 1 μL miRNA, 1 μL 1 nmol / L SLP, 1 μL 1 nmol / L SLP-pam, and 0.6 μL 10× SplintR ligase buffer. The final 6 μL mixture consists of 2.4 μL rnase-free water. Incubate at 85°C for 2 minutes, then at 37°C for 5 minutes. Add 0.1 μL 25 U / μL SplintR ligase, 0.4 μL 10×SplintR ligase reaction buffer, and 3.5 μL rnase-free water, and incubate at 37°C for 15 minutes to obtain the ligase product. Then, add 2 μL of ligand to the LAMP amplification system, including 0.5 μL 8 U / μL Bst 2.0 WarmStart DNA polymerase, 1.0 μL 10× ThermoPol reaction buffer, and 0.4 μL 20 μmol / L FIP. 0.4 μL of 20 μmol / L LBIP, 1.0 μL of 2.5 mmol / L dNTPs, and 2.7 μL of RNase-free water were added. The mixture was reacted at 65°C for 20 minutes. Finally, a CRISPR / Cas12a reaction mixture consisting of 0.2 μL of 10 μM Cas12a nuclease, 2 μL of 10× NE Buffer 2.1, 1 μL of 10 μmol / L ssDNA reporter gene, 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) was added. The mixture was then stored at 37°C for 60 minutes. After the reaction was complete, the fluorescence value was observed.

[0038] This method requires only a pair of stem-loop DNA probes with two key components—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, catalyzing the formation of the double stem-loop DNA structure. These ligation products then initiate a LAMP reaction. After the LAMP reaction, 2 µL of the amplification product is added to a CRISPR-Cas12a reaction system, and fluorescence intensity is observed at 37°C for 60 min.

[0039] Sensitivity test results 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 two other target miRNAs (novel-miR574 and novel-miR248). NTC2 is a system for detecting novel-miR574, used to detect the other two target miRNAs (novel-miR248 and novel-miR544). NTC3 is a system for detecting novel-miR248, used to detect the other two target miRNAs (novel-miR544 and novel-miR574). Only the target miRNA can act as a splice to ligate its corresponding specific stem-loop DNA probe, initiating subsequent LAMP and producing a clear fluorescent signal. In contrast, in the presence of other miRNAs, the specific stem-loop DNA probe cannot ligate effectively, and due to the lack of a matching splice template and subsequent LAMP amplification, CRISPR-Cas12a has no target to recognize.

[0044] Three target primers (Table 1) were used to detect miRNAs on needles from inoculated Korean pine nematodes and control groups, respectively. Only novel-miR574 was identified in the samples, which is 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 untreated Korean pine, Korean pine treated with water, and Korean pine inoculated with pine wilt disease, the nematode-inoculated sample showed significant fluorescence on day 10 after inoculation, while the untreated sample and the untreated Korean pine showed no fluorescence. In conclusion, novel-miR574 can serve as an early diagnostic target for pine wilt disease in Korean pine.

Claims

1. The application of novel-miR574 as a diagnostic target for whether Korean pine is infected with pine wilt disease includes: The expression level of novel-miR574 in the sample was detected by CAL-LAMP method, wherein the nucleotide sequence of novel-miR574 is shown in SEQ ID No. 13.

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