RPA-CRISPR / Cas12b system-based visual detection kit for phaseohagi phytophthora blight and application of RPA-CRISPR / Cas12b system-based visual detection kit

Abstract

The invention relates to the technical field of kit detection, in particular to an RPA-CRISPR / Cas12b system-based visual detection kit for phaseolospora vibriosa and application of the visual detection kit for phaseolospora vibriosa based on the RPA-CRISPR / Cas12b system. The primer group disclosed by the invention comprises an RPA (recombinase polymerase amplification) primer pair and SgRNA (single guide ribonucleic acid), the RPA amplification primer pair comprises an upstream primer as shown in SEQ ID NO. 7 and a downstream primer as shown in SEQ ID NO. 8; and the SgRNA is as shown in SEQ ID NO. 26. And a basis is provided for rapid detection of phaseolospora piricola.

Description

Technical Field

[0001] This invention relates to the field of reagent kit detection technology, and in particular to a visual detection kit for Phytophthora beanus based on the RPA-CRISPR / Cas12b system and its application. Background Technology

[0002] Common bean halo blight ( Pseudomonas savastanoi pv.phaseolicola Psph (Psphate of Plant Bean) is a quarantine pathogen, and even 0.02% seed infection can cause disease outbreaks. Current molecular biological detection methods for Psphate of Plant Bean include PCR, duplex PCR, real-time fluorescent PCR, loop-mediated isothermal amplification (LAMP), lock-probe technology, and droplet digital PCR. While PCR is a mature technology for detecting plant pathogens, it requires thermal cycling (temperatures above 94°C) and has a long reaction time, making it unsuitable for rapid on-site detection. LAMP amplifies nucleic acids isothermally at 60-65°C, but requires high temperatures, necessitates large-capacity batteries to maintain temperature control, suffers from significant false positives, and demands sophisticated primer design.

[0003] Current rapid detection technologies for Phytophthora beanus have limitations in terms of accurate identification and specificity. Therefore, more accurate detection methods for Phytophthora beanus need to be studied.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a visual detection kit for Phytophthora bean blight based on the RPA-CRISPR / Cas12b system and its application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a primer set for detecting common bean blight pathogens based on the RPA / CRISPR-Cas12b system, including RPA amplification primer pairs and SgRNA; The RPA amplification primer pair includes the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8; The SgRNA is shown in SEQ ID NO.26.

[0007] The present invention also provides the application of the primer set described herein in the preparation of products that specifically detect Phytophthora beanus.

[0008] Preferably, the product includes detection reagents, detection kits, molecular markers, or molecular probes.

[0009] The present invention also provides a kit for detecting Phytophthora chinensis of common bean, comprising the aforementioned primer set.

[0010] The present invention also provides the application of the kit in the specific detection of Phytophthora beanus.

[0011] The present invention also provides a method for detecting *Phytophthora chinensis* of common bean using the aforementioned primer set, comprising the following steps: (1) Extract DNA from the sample to be tested to obtain template DNA; (2) Amplify the template DNA obtained in step (1) using RPA amplification primers to obtain the amplification product; (3) Mix SgRNA, AaCas12b enzyme and the amplification product described in step (2) and perform CRISPR-Cas12b reaction; (4) Visualize the results of the reaction.

[0012] Preferably, the amplification system in step (2) is: 29-30 µL of Rehydration Buffer (2×), 2-3 µL of Magnesium Acetate Solution, 1.5-2.5 µL of each upstream and downstream primer in the RPA amplification primer pair, 1.5-2.5 µL of Template DNA, and ddH2O to bring the total to 50 µL. The concentration of magnesium acetate solution is 260~300 mmol / L; The concentrations of the upstream and downstream primers were independently 9–11 µmol / L.

[0013] Preferably, the reaction system for the CRISPR-Cas12b reaction in step (3) is as follows: 1.5-2.5 μL of 2-3 μmol / L AaCas12b enzyme, 4.5-5.5 μL of 10×AaCas12b buffer, 0.7-1.3 μL of 2.5-3.5 μmol / L SgRNA, 0.4-0.6 μL of fluorescent reporter molecule, 0.8-1.2 μL of amplification product, and ddH2O to bring the total volume to 50 µL. The concentration of the fluorescent reporter molecule is 200~2000 nmol / L.

[0014] Preferably, the fluorescent reporter molecule is 5'-FAM-TTTTTTT-BHQ1-3' or 5'-Biotin-TTTTTTT-FAM-3'.

[0015] The present invention also provides the application of the method in the specific detection of Phytophthora beanus.

[0016] The present invention has the following advantages: This invention targets the pathogen of *Phytophthora haliotis*, by designing specific recombinase polymerase to amplify RPA primer pairs and CRISPR-Cas12b-based SgRNA, establishing an isothermal amplification method combining RPA and CRISPR-Cas12b. Compared to conventional PCR / q-PCR, this method offers higher sensitivity and better specificity, while significantly reducing detection time to 40 minutes. The method provided by this invention is simple, time-efficient, highly sensitive, and specific, conducted entirely at 37°C, requiring minimal equipment and easily implemented in the field, demonstrating excellent application potential and providing reliable technical support for the prevention and control of *Phytophthora haliotis*. Attached Figure Description

[0017] Figure 1 For the optimization of amplification conditions (A is the primer screening result, B is the SgRNA screening result, and C is the optimal result of the fluorescent reporter molecule concentration); Figure 2 This represents the sensitivity detection results of the fluorescence method; Figure 3 The results are from the sensitivity test of the lateral flow chromatography test strip; Figure 4 The results of fluorescence detection of *Phytophthora beanus* in infected pea seeds; Figure 5 This study aims to detect the results of lateral flow chromatography test strips in the detection of *Phytophthora beanus* causal agent in infected pea seeds. Detailed Implementation

[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] The common bean halo blight pathogen described in this embodiment of the invention is LMG 5475, derived from the Belgian Co-ordinated Collections of Microorganisms.

[0020] In this embodiment of the invention, fluorescent reporter molecule 1 is 5'-FAM-TTTTTTTT-BHQ1-3'; fluorescent reporter molecule 2 is biotin-labeled, specifically 5'-Biotin-TTTTTTT-FAM-3'.

[0021] Example 1

[0022] Screening of RPA amplification primers

[0023] We screened the published gene sequences of *Phytophthora beanense* causal agent in the NCBI database (National Center for Biotechnology Information) and selected the gene encoding bean toxin resistance. argK-tox Using the partial sequence (Gene Bank: AB237164.1) as the primer design object, specific primers that meet the RPA reaction conditions were designed, and 12 pairs of RPA primers were designed (Table 1) (RPA-F1 / R1 to RPA-F12 / R12).

[0024] In the RPA amplification products, the protospacer adjacent motif (PAM) was searched. Based on the PAM sequence position, SgRNAs (4 pairs) that are complementary to the target sequence bases were designed. The primer pairs and SgRNAs are shown in Table 1 and were synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd.

[0025] Table 1 RPA Primer Set

[0026] Example 2

[0027] Amplification conditions optimization

[0028] 1. Primer and SgRNA screening

[0029] Primer selection is crucial for RPA amplification efficiency, while SgRNA cleavage efficiency is a significant factor affecting Cas12b detection efficiency. Therefore, primer selection for the coding gene ( argK-tox Twelve primer pairs and four SgRNAs were screened. The screening results for primer pairs and SgRNAs are as follows: Figure 1 As shown. To screen for the optimal primer and SgRNA combination for the gene, SgRNA (1-4) was used on 12 primer pairs (F1 / R1~F12 / R12), and the best-performing primer pairs F4 / R4 (SEQ ID NO. 7~8) and SgRNA-2 (SEQ ID NO. 26) were identified for subsequent experiments. Figure 1 A and Figure 1 B).

[0030] 2. Optimization of Fluorescent Reporter Molecule Concentration: The fluorescent reporter molecule was optimized to final concentrations of 200 nmol / L, 400 nmol / L, 600 nmol / L, 800 nmol / L, 1000 nmol / L, and 2000 nmol / L. Fluorescence values ​​were collected at 495 nm wavelength and 37℃ for 40 min using a real-time quantitative PCR instrument, as referenced. Figure 1 C. According to the fluorescence results, the fluorescence signal was strongest when the reporter molecule concentration was 2000 nmol / L. As the reporter molecule concentration decreased, the fluorescence signal weakened, reaching a weak point at 200 nmol / L. Therefore, considering experimental cost, 400 nmol / L was chosen as the optimal concentration for this experiment.

[0031] 3. RPA amplification reaction

[0032] The template DNA was mixed with the RPA system to rapidly amplify the target nucleic acid sequence. The specific RPA reaction system was prepared as follows: 29.5 µL of Rehydration Buffer (2×), 2.5 µL of magnesium acetate solution (280 mmol / L), 2 µL each of 10 μmol / L RPA primers RPA-F4 / RPA-R4, 2 µL of template DNA, and ddH2O were added to a centrifuge tube to bring the volume to 50 µL. After mixing, the tube was placed in a portable heating device and incubated at 37 °C for 20 min to obtain the amplification product.

[0033] Template DNA: Obtained after extracting DNA from the sample to be tested.

[0034] 4. CRISPR-Cas12b reaction

[0035] (1) CRISPR-Cas12b fluorescence detection: The amplification product was mixed with the CRISPR-Cas12b reaction system to rapidly amplify the target nucleic acid sequence. The specific ratio was as follows: 2 μL of 2.5 μmol / L AaCas12b enzyme, 5 μL of 10×AaCas12b buffer, 1 μL of 3 μmol / L SgRNA, 0.5 μL of 400 nmol / L fluorescent reporter molecule 1, 1 μL of amplification product, and ddH2O to bring the total volume to 50 µL. After thorough mixing, the mixture was reacted at 45 °C for 15 min in a heated metal bath. Visual detection was performed using a portable blue light spectrometer (450 nm). When the solution was observed to emit green fluorescence with the naked eye, the reaction result was considered positive. The SgRNA was SgRNA-2 as shown in SEQ ID NO.26.

[0036] (2) CRISPR-Cas12b lateral flow chromatography test strip detection: The amplification product was mixed with the CRISPR-Cas12b lateral flow chromatography test strip system to rapidly amplify the target nucleic acid sequence. The system ratio was: 2 μL AaCas12b enzyme (2.5 μmol / L), 5 μL 10×AaCas12b Buffer, 1 μL SgRNA (3 μmol / L), 0.5 μL 400 nmol / L fluorescent reporter molecule 2, 1 μL amplification product, and ddH2O to a final volume of 50 µL. The mixture was incubated at 45 °C for 15 min. After the reaction was complete, 50 µL of ddH2O was added, and the mixture was thoroughly mixed. The sample end of the test strip was then immersed in the solution for 3 min, and the T line (detection band) was observed for color development to determine the detection result. The SgRNA was SgRNA-2 as shown in SEQ ID NO.26.

[0037] Example 3

[0038] Sensitivity detection

[0039] DNA (163 ng / μL) from the standard strain of common bean halo blight (LMG 5475) was used as a template and diluted to DNA concentrations of 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL for sensitivity detection. ddH2O was used as a negative control. Fluorescence and lateral flow chromatography test strip sensitivity experiments were conducted.

[0040] The detection methods for fluorescence and lateral flow chromatography test strips are as shown in Example 2. Sensitivity detection results are as follows: Figures 2-3 As shown.

[0041] Figure 2 As shown, CK is the negative control. Green light can be observed under blue light at concentrations of 100 ng / μL, 10 ng / μL, 1 ng / μL, and 100 pg / μL, but only a weak green light is observed at 10 pg / μL, making it difficult to distinguish. This indicates that the sensitivity of the RPA / CRISPR-Cas12b fluorescence method is 10 pg / μL. Figure 3 As shown, CK serves as the negative control. Standard strains were serially diluted 10-fold to test the sensitivity of the CRISPR-Cas12b lateral flow chromatography test strip system. The detection limit of the second strip from the left is 10 ng / μL, at which concentration of DNA a very faint T-line (detection band) appears. Sensitivity testing revealed that the detection limit of both the fluorescence detection system and the lateral flow chromatography test strip system was 10 pg / μL, with results obtained in 40 minutes for both systems. The sensitivity of the RPA / CRISPR-Cas12b fluorescence detection system is comparable to that of quantitative qPCR, and the fluorescence detection system is even more sensitive than qPCR, while qPCR is more sensitive than conventional PCR. For higher detection sensitivity, CRISPR-Cas12b fluorescence detection can be used; for more convenient on-site testing, CRISPR-Cas12b lateral flow chromatography test strips can be used.

[0042] Example 4

[0043] Simulated on-site sample testing and application

[0044] Healthy pea seeds, confirmed to be free from bean blight pathogen infection through quarantine, were selected as the sample matrix. After washing with sterile water, the seeds were completely immersed in a solution with a concentration of 1×10⁻⁶. 7The *Lactobacillus lentigines* (LMG 5475) bacterial suspension (CFU / mL) was incubated for 2 hours, then aseptically air-dried for 2 days and stored at 4°C for later use. In the simulated sample detection experiment, two infected seeds were mechanically ground and pulverized, then 1 mL of TE buffer was added and allowed to stand for 5 minutes. 500 μL of the supernatant was then heated in a 95°C metal bath for 10 minutes to lyse and release nucleic acids. 2 μL of the lysate supernatant was used as a template. The optimized RPA / CRISPR-Cas12b detection system from Example 2 was used, and detection was performed using both fluorescence and lateral flow chromatography strip methods. Each sample was tested in triplicate. The experimental results are as follows: Figures 4-5 As shown, all six simulated infected seed samples exhibited a clear green fluorescent signal in the fluorescence method and a clear detection line (T line) in the test strip method. The results of the two detection methods were completely consistent, with a positive detection rate of 100%. This indicates that the RPA / CRISPR-Cas12b detection system established in this invention has high detection accuracy and repeatability against *Phytophthora chinensis*, and can effectively identify and detect the target pathogen in simulated infected seed samples.

[0045] As can be seen from the above embodiments, the RPA / CRISPR-Cas12b detection method provided by the present invention has outstanding advantages such as rapid detection, intuitive result interpretation, simple operation, and no reliance on complex instruments and equipment. It is suitable for rapid screening and early diagnosis in field, port quarantine and other scenarios, providing reliable technical support for the timely prevention and control of this disease.

Claims

1. A primer set for detecting *Phytophthora chinensis* var. *comfortella* based on the RPA / CRISPR-Cas12b system, characterized in that... Including RPA amplification primer pairs and SgRNA; The RPA amplification primer pair includes the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8; The SgRNA is shown in SEQ ID NO.

26.

2. The application of the primer set according to claim 1 in the preparation of products specifically for detecting Phytophthora beanus.

3. The application according to claim 2, characterized in that, The products include detection reagents, detection kits, molecular markers or molecular probes.

4. A kit for detecting Phytophthora blight in common bean, characterized in that, Includes the primer set as described in claim 1.

5. The use of the kit according to claim 4 in the specific detection of Phytophthora beanus.

6. A method for detecting *Phytophthora chinensis* var. *chinensis* using the primer set described in claim 1, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested to obtain template DNA; (2) Amplify the template DNA obtained in step (1) using RPA amplification primers to obtain the amplification product; (3) Mix SgRNA, AaCas12b enzyme and the amplification product described in step (2) and perform CRISPR-Cas12b reaction; (4) Visualize the results of the reaction.

7. The method according to claim 6, characterized in that, The amplification system described in step (2) is as follows: 29-30 µL of Rehydration Buffer (2×), 2-3 µL of Magnesium Acetate Solution, 1.5-2.5 µL of each upstream and downstream primer in the RPA amplification primer pair, 1.5-2.5 µL of Template DNA, and ddH2O to bring the total to 50 µL. The concentration of magnesium acetate solution is 260~300 mmol / L; The concentrations of the upstream and downstream primers were independently 9–11 µmol / L.

8. The method according to claim 6, characterized in that, The reaction system for the CRISPR-Cas12b reaction in step (3) is as follows: 1.5-2.5 μL of 2-3 μmol / L AaCas12b enzyme, 4.5-5.5 μL of 10×AaCas12b buffer, 0.7-1.3 μL of 2.5-3.5 μmol / L SgRNA, 0.4-0.6 μL of fluorescent reporter molecule, 0.8-1.2 μL of amplification product, and ddH2O to bring the total volume to 50 µL. The concentration of the fluorescent reporter molecule is 200~2000 nmol / L.

9. The method according to claim 8, characterized in that, The fluorescent reporter molecule is 5'-FAM-TTTTTTT-BHQ1-3' or 5'-Biotin-TTTTTTT-FAM-3'.

10. The application of the method according to any one of claims 6 to 9 in the specific detection of Phytophthora beanus.

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