A primer composition for detecting a pathogen of rose branch blight and application thereof

Abstract

This invention discloses a primer composition for detecting the pathogen of rose twig blight and its application, belonging to the field of microbial detection technology. This invention targets the pathogen of rose twig blight. Cytospora lhasaensis Specific primers were designed based on the ITS sequence. The 5' end of the downstream primer was labeled with biotin, and a 46-52 bp NFO probe was added to the reaction system. The 5' end of this probe was labeled with FAM, the 30th bp from the 5' end was replaced with THF, and polymerase extension was added to the 3' end to block the gene. Using this primer, specific primers can be used to target the ITS sequence. Cytospora lhasaensis Its rapid, sensitive, and specific detection capabilities can be used in various situations such as field testing, seedling quarantine, and import / export plant inspection at ports, and it has significant value for widespread application.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to a primer composition for detecting the pathogen of rose twig blight and its application. Background Technology

[0002] rose( Rosa spp. Rose is one of the world's most important ornamental flowers, with significant economic value in landscaping, cut flower production, and the fragrance industry. Rose twig blight is one of the major diseases affecting rose production, and its pathogen (…) Cytospora lhasaensis This disease can infect the stems and branches of roses, causing browning and death of the branches, leaf drop, and in severe cases, death of the entire plant, resulting in huge economic losses to the flower industry. The disease is particularly severe under high humidity and warm conditions, and it spreads easily through pruning tools, air currents, and rainwater, making it difficult to control.

[0003] Currently, the main methods for detecting the pathogen of rose twig blight include the following: Traditional morphological identification: This method relies on the colony morphology, conidia, and microscopic observation of the pathogen. It is cumbersome, time-consuming (usually 3-7 days), and requires highly skilled personnel, making it unsuitable for large-scale rapid field screening. Conventional PCR and real-time quantitative PCR (qPCR): These methods offer high specificity and sensitivity, but both rely on sophisticated thermal cycling equipment, demanding strict laboratory conditions and skilled personnel. The detection process is complex, typically taking several hours, and the equipment is inconvenient to carry, making it difficult to meet the practical needs of field and port-based testing. The common shortcomings of these methods are: long detection cycles, reliance on specialized instruments, complex operation, and unsuitability for point-of-care testing (POCT), thus failing to effectively support early warning and rapid control of the disease. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a primer composition for detecting the pathogen of rose twig blight and its application, thereby achieving rapid, sensitive, and specific detection of the pathogen, shortening the detection cycle, and improving detection efficiency and accuracy.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method for detecting the pathogen of rose twig blight is provided. Cytospora lhasaensis The primer composition includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2.

[0006] This invention provides a primer composition as described above for the preparation of a primer for detecting the pathogen of rose twig blight. Cytospora lhasaensis Application in reagent kits.

[0007] This invention provides a method for detecting the pathogen of rose twig blight. Cytospora lhasaensis The PCR detection kit includes the primer composition described above.

[0008] This invention provides a method for detecting the pathogen of rose twig blight. Cytospora lhasaensis The RAA-LFD rapid detection kit includes the primer composition described above.

[0009] Furthermore, the RAA-LFD rapid detection kit also includes an LFD probe, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0010] Furthermore, the 5' end of the LFD probe is labeled with a FAM fluorescent group, and the 3' end is connected to a C3 spacer blocking group.

[0011] Furthermore, the RAA-LFD rapid test kit also includes a lateral flow chromatography test strip and an LFD detection buffer; the lateral flow chromatography test strip includes an anti-FAM colloidal gold marker, a test line, and a control line.

[0012] This invention provides the above-mentioned RAA-LFD rapid detection kit for detecting the pathogen of rose twig blight. Cytospora lhasaensis Applications in [the context of the text].

[0013] This invention provides a pathogen of rose twig blight. Cytospora lhasaensis The detection method includes the following steps: (1) Extract total DNA from the diseased tissue of the rose plant to be tested; (2) Using the total DNA from step (1) as a template, perform RAA amplification using the RAA-LFD rapid detection kit according to any one of claims 4-7; (3) Add LFD detection buffer to the RAA amplification product from step (2), then insert the lateral flow chromatography strip, incubate at room temperature, and then visually interpret the results.

[0014] Furthermore, the criteria for determining the test result are as follows: if both the test line and the control line of the lateral flow chromatography test strip are colored, the result is positive; if only the control line of the lateral flow chromatography test strip is colored, the result is negative; if the control line of the lateral flow chromatography test strip is not colored, the result is invalid.

[0015] The present invention has the following beneficial effects: ① Fast detection speed: The entire detection process takes no more than 60 minutes (DNA extraction takes about 30 minutes, RAA amplification takes 20-30 minutes, and LFD interpretation takes 2 minutes), which is far superior to traditional morphological identification (3-7 days) and PCR method (4-6 hours).

[0016] ② High primer specificity: The designed primers and probes target the ITS region of the rose twig blight pathogen and do not cross-react with other common plant pathogens.

[0017] ③ High sensitivity: The detection sensitivity can reach 1 ng / μL template DNA level.

[0018] ④ Easy to operate: The RAA reaction does not require a thermal cycler, but can be completed with a constant temperature water bath or portable heating device; the LFD results can be read by the naked eye and no fluorescence detection instrument is required.

[0019] ⑤ Wide range of applications: The primers of this invention can be used to prepare lateral flow test strips coated with biotin and FAM antibodies, which can be used in various occasions such as field testing, seedling quarantine, and port inspection of imported and exported plants, and have important value for promotion and application. Attached Figure Description

[0020] Figure 1 The results of screening RAA primers for the rose twig blight pathogen in PCR reaction in Example 2 of this invention; Figure 2 The results of isothermal amplification electrophoresis gel screening of RAA primers for the pathogen of rose twig blight in Example 2 of this invention; Figure 3 This is the optimization result of the RAA reaction electrophoresis visualization system for the rose twig blight pathogen in Example 2 of the present invention; Figure 4 The pathogenic fungus RAA of rose twig blight in Example 2 of this invention Optimization results of final concentration of LFD probe reaction; Figure 5 The pathogenic fungus RAA of rose twig blight in Example 2 of this invention Results of LFD specificity study; Figure 6 The pathogenic fungus RAA of rose twig blight in Example 2 of this invention Results of LFD sensitivity study. Detailed Implementation

[0021] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] Example 1: Specific Primer Design This invention first screens for molecular detection targets of the pathogen causing rose twig blight. Studies have shown that the internal transcribed spacer (ITS) region of ribosomal DNA exhibits high sequence variability among fungal species, while remaining relatively conserved within species. It is a widely used and recognized reliable molecular marker for the molecular identification and detection of plant pathogenic fungi. Therefore, this invention selects the ITS sequence (as shown in SEQ ID NO. 6) as the target gene for the specific detection of rose twig blight. To obtain accurate target gene sequence information, this invention isolates and purifies pathogenic fungal strains from typical rose twig blight samples, extracts their genomic DNA as a template, and uses the universal fungal primers ITS1 / ITS4 to amplify the target fragment by PCR and sequence it. The sequenced sequences are submitted to the GenBank database of the National Center for Biotechnology Information (NCBI) and homology search and alignment analysis are performed using the BLAST (Basic Local Alignment Search Tool) online alignment tool. Specifically, on the NCBI BLAST homepage, select the "Nucleotide BLAST" function, input the ITS sequence obtained from sequencing, set the alignment database to "nt (non-redundant nucleic acid sequence database)," select the "Highly similar sequences (megablast)" alignment mode, and perform homology sequence search on all known sequences in the database. The BLAST alignment results show that the ITS sequence obtained in this invention is similar to the reported rose twig blight pathogens (such as...) in the database. Neofusicoccum parvum , Lasiodiplodia theobromae The corresponding ITS sequence (etc.) showed extremely high homology, indicating that the sequence is indeed the ITS gene fragment of the pathogen of rose twig blight. The above sequence alignment results not only verified the taxonomic position of the isolated strain but also provided a reliable sequence basis for the subsequent design of specific primers and probes. Based on these results, two upstream primers and two downstream primers were designed, forming four primer pairs, as detailed in Table 1.

[0023] The amplification performance of each primer pair was initially verified using conventional PCR. Specifically, using the target nucleic acid sequence as a template, PCR amplification was performed using the designed candidate primers. Agarose gel electrophoresis showed that all four primer pairs exhibited specific amplification bands at the expected positions, indicating that all candidate primers possessed basic amplification capabilities. The conventional PCR reaction system was as follows: PCR Mix 10.0 µL, upstream primer (10 μM) 1.0 µL, downstream primer (10 μM) 1.0 µL, purified water 7.0 µL, gDNA 1.0 µL, for a total reaction volume of 20.0 µL. The reaction program was: 95 °C pre-denaturation for 3 min, 95 °C denaturation for 30 s, 56 °C annealing for 30 s, 72 °C extension for 1 min (30 cycles from denaturation to extension), 72 °C complete extension for 1 min, and storage of PCR products at 4 °C.

[0024] Table 1 Primer Sequences

[0025] Example 2: Optimization of RAA Reaction System and Conditions To further screen for the optimal primer pairs suitable for RAA-LFD detection, this invention applies the above four primer pairs to the RAA reaction system. An LFD probe CI-RAA-P is designed for the RAA reaction system, with the following sequence (5'-3'): [FAM]CGAAATGCGATAAGTAATGTGAATTGCAGA[THF]ATTCAGTGAATCATCG[C3 Spacer]; Wherein, FAM: the 5' end of the probe is marked with 6 Carboxyfluorescein; THF: Tetrahydrofuran, the 30th position of the shown nucleotide is modified with THF as the cleavage site for the nuclease nfo; C3 Spacer: the 3' end of the probe is modified with C3 Spacer. (When creating the sequence listing, the modifying groups FAM, THF, and C3 Spacer are deleted, and the modified sequence is as follows: CGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCG, SEQ ID NO.5) The primers were subjected to isothermal amplification at 25℃ for 30 min, and the amplification products were detected by agarose gel electrophoresis. The total volume of the RAA reaction was 50 μL, with the following composition: lyophilized RAA reaction powder (TwistAmp® exo or equivalent domestic RAA kit): 1 tube; RAA resuspension buffer: 25.0 μL; upstream primer (10 μmol / L): 2.0 μL; downstream primer (10 μmol / L): 2.0 μL; DNA template: 2 μL; sterile double-distilled water: 14 μL; magnesium acetate solution (280 mmol / L): 5 μL (added to the cap last, inverted to mix, and the reaction started). By comparing the amplification efficiency, specificity, and product yield of each primer pair, the primer pair with the strongest amplification signal and the fewest non-specific bands, namely primer pair 4 (CI-RAA-F2 / CI-RAA-R2), was finally selected as the optimal primer pair (see [link to product description]). Figure 2 This is used for the subsequent optimization of the RAA testing system and the establishment of methods.

[0026] After optimizing the design of RAA primers and probes, this invention first explored and optimized the RAA detection system to determine the optimal reaction system. Preliminary experiments revealed that reaction temperature, reaction time, and final primer concentration significantly affected the experimental results. Therefore, reaction temperature, reaction time, and final primer concentration were considered as influencing factors for the RAA reaction, and horizontal gradients were designed: reaction temperature gradients of 5, 15, 25, 30, 37, and 40°C; reaction time gradients of 5, 10, 15, 20, 30, and 40 min; and final primer concentrations of 200, 400, 600, and 800 nM. Initially, with reaction temperature as the variable, the reaction time was uniformly set to 30 min, and the final primer concentration was set to 400 nM. After identifying the optimal reaction temperature, the optimal reaction time was explored, and finally, the optimal final primer concentration was determined. The specific procedures are as follows: Reaction temperature gradients were set to 5, 15, 25, 30, 37, and 40℃; reaction time gradients were set to 5, 10, 15, 20, 30, and 40 min; and final primer concentration gradients were set to 200, 400, 600, and 800 nM. Optimization was performed in the following order: First, the reaction time was fixed at 30 min and the final primer concentration at 400 nM, using the amplification product yield as an indicator to screen for the optimal reaction temperature; then, using this optimal temperature condition, the final primer concentration was fixed at 400 nM, and the optimal reaction time was screened; finally, the optimal final primer concentration was screened at both the optimal temperature and optimal time. Each gradient experiment was repeated three times to ensure the reliability of the results.

[0027] Depend on Figure 3It was found that in the RAA reaction assay of the pathogen of rose twig blight, under a fixed reaction time of 30 min, the RAA amplification products at different temperatures were detected by agarose gel electrophoresis. The electrophoresis results showed that at room temperature (25℃), the target band was clear, and there were no primer dimers or non-specific bands in the lanes, indicating that the amplification efficiency and specificity of the RAA reaction were suitable at this temperature, thus determining 25℃ as the optimal reaction temperature. With the reaction temperature fixed at 25℃, seven time gradients were set, and the accumulation of products at different reaction times was compared by gel electrophoresis. Electrophoresis results showed that within the range of 5-20 min, the brightness of the target band gradually increased with the extension of reaction time, indicating a continuous increase in the amount of amplified product. At 30 min, the band brightness reached its peak, with uniform width and a clean background, free of tailing or stray bands, indicating that amplification had entered a plateau phase and the product amount had reached its maximum. Further extending the reaction time to 40 min did not show a significant increase in band brightness; instead, some lanes exhibited slight diffusion or primer dimer bands. This may be due to decreased enzyme activity or increased non-specific side reactions caused by excessively long reaction times. Therefore, to ensure the highest product yield and optimal specificity while also considering detection efficiency, 30 min was selected as the optimal reaction time. By comparing the amplification efficiency, specificity, and product yield of the RAA reaction at different primer final concentrations, the primer concentration with the strongest amplification signal was finally selected as the optimal final primer concentration. When the final primer concentration was 600 nM, the RAA reaction amplification efficiency was the highest, and the bands were clear and uniform. Therefore, the preferred final primer concentration was 600 nM for subsequent optimization of the RAA detection system and method establishment.

[0028] After optimizing the basic RAA reaction system (temperature, time, and primer concentration), this invention further optimizes the final probe concentration using a single-factor approach to improve the overall performance of the RAA-LFD detection method. Specifically, under optimal temperature, optimal reaction time, and optimal primer concentration conditions, final probe concentrations of 120, 200, 300, and 400 nM were set. The intensity of the colorimetric signal on the detection line of the side-flow chromatography strip was used as the evaluation index to screen for the optimal probe concentration, which was then used for subsequent evaluation experiments on sensitivity, specificity, and actual sample detection. In the final probe concentration optimization experiment, four gradients of 120, 200, 300, and 400 nM were set for the reaction.

[0029] The results showed that when the final probe concentration was 120 nM, the LFD test strip detection line exhibited a clear and bright colored band with a clean background and no non-specific signal; when the concentration increased to 200 nM and above, the signal intensity of the detection line did not show a significant increase (see...). Figure 4Taking into account signal intensity, background cleanliness, and detection cost, this invention determines the optimal final concentration of the probe in the RAA-LFD detection system to be 120 nM. The specific composition of the final RAA-LFD reaction is as follows: lyophilized RAA reaction powder (TwistAmp®exo or equivalent domestic RAA kit): 1 tube; RAA resuspension buffer: 25 μL; upstream primer (10 μmol / L): 3 μL; downstream primer RAA-R (10 μmol / L): 3 μL; LFD probe Probe (10 μmol / L): 0.6 μL; DNA template: 2 μL; sterile double-distilled water: 11.4 μL; magnesium acetate solution (280 mmol / L): 5 μL (add to the tube cap last, invert to mix, and start the reaction).

[0030] Add 5 μL of RAA amplification product to 45 μL of LFD detection buffer, mix thoroughly, insert the LFD test strip vertically into the mixture, let it stand at room temperature (25℃) for 2 minutes, then remove the test strip and observe the result with the naked eye.

[0031] Example 3: Specificity and Sensitivity Detection of Primer Compositions Using gDNA from closely related pathogenic fungi and other pathogenic fungi of the same genus preserved in our laboratory as templates (see Table 2), RAA-LFD detection was performed to evaluate the specificity of the primer composition screened in this invention. Figure 5 It can be seen that, when the primer composition of the present invention is used for detection, the pathogen of rose twig blight can be identified. Cytospora lhasaensis The test results showed no cross-reactivity with other pathogenic fungi, indicating that the primer composition of the present invention has good specificity.

[0032] Table 2. Pathogenic fungi involved in specific detection

[0033] The gDNA stock solution of the rose twig blight pathogen was serially diluted and used as a template, with a water control included. Amplification was performed under optimal conditions for the RAA-LFD identification method, and the results are as follows: Figure 6 As shown. By Figure 6 It is evident that all test strips have control lines, proving the validity of the test results. From a gDNA concentration of 1 ng / µL, the test line gradually darkens with increasing concentration. Therefore, the RAA of rose twig blight provided by this invention... The LFD identification method has a detection limit of 1 ng / µL for gDNA.

[0034] The ITS sequence of the pathogen in this invention is as follows: (SEQ ID NO.6).

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting the pathogen of rose twig blight. Cytospora lhasaensis The primer composition is characterized by, The primer composition includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1; and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

2.

2. The primer composition of claim 1 is used in the preparation of a primer for detecting the pathogen of rose twig blight. Cytospora lhasaensis Application in reagent kits.

3. A method for detecting the pathogen of rose twig blight. Cytospora lhasaensis The PCR detection kit is characterized by, The PCR detection kit includes the primer composition of claim 1.

4. A method for detecting the pathogen of rose twig blight. Cytospora lhasaensis The RAA-LFD rapid detection kit is characterized by, The RAA-LFD rapid detection kit includes the primer composition as described in claim 1.

5. The RAA-LFD rapid detection kit according to claim 4, characterized in that, The RAA-LFD rapid detection kit also includes an LFD probe, the nucleotide sequence of which is shown in SEQ ID NO.

5.

6. The RAA-LFD rapid detection kit according to claim 5, characterized in that, The LFD probe is labeled with a FAM fluorescent group at its 5' end and connected to a C3 spacer blocking group at its 3' end.

7. The RAA-LFD rapid detection kit according to claim 4, characterized in that, The RAA-LFD rapid detection kit also includes a lateral flow chromatography test strip and an LFD detection buffer; the lateral flow chromatography test strip includes an anti-FAM colloidal gold marker, a detection line, and a control line.

8. The RAA-LFD rapid detection kit according to any one of claims 4-7 for detecting the pathogen of rose twig blight. Cytospora lhasaensis Applications in [the context of the text].

9. A pathogen of rose twig blight Cytospora lhasaensis The detection method is characterized by, Includes the following steps: (1) Extract total DNA from the diseased tissue of the rose plant to be tested; (2) Using the total DNA from step (1) as a template, perform RAA amplification using the RAA-LFD rapid detection kit according to any one of claims 4-7; (3) Add LFD detection buffer to the RAA amplification product from step (2), then insert the lateral flow chromatography strip, incubate at room temperature, and then visually interpret the results.

10. The detection method according to claim 9, characterized in that, The criteria for the determination of the detection method are as follows: if both the test line and the control line of the lateral flow chromatography test strip are colored, the result is positive; if only the control line of the lateral flow chromatography test strip is colored, the result is negative; if the control line of the lateral flow chromatography test strip is not colored, the result is invalid.

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