Rapid detection method for Xanthomonas oryzae pv. Oryzae based on RPA-CRISPR / Cas12b technology

Abstract

The invention discloses a rapid detection method for Xanthomonas oryzae pv. Oryzae on the basis of RPA-CRISPR / Cas12b. The reagent combination comprises a specific RPA primer pair and sgRNA, and the sequences of the specific RPA primer pair and the sgRNA are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. The detection method comprises the following steps: taking nucleic acid of a sample to be detected as a template, and performing RPA isothermal amplification by utilizing the primer pair; the amplification product is mixed with sgRNA, Cas12b protein and fluorescent reporter molecules for incubation, and the trans-cleavage activity of Cas12b is activated; a result is judged by detecting a fluorescence signal. According to the invention, the optimized RPA and CRISPR systems are integrated on the centrifugal micro-fluidic chip, so that the full-flow closed tube and automatic detection is realized, the detection can be completed within 30 minutes, the sensitivity reaches 0.156 pg / reaction, no non-specific reaction is caused to five related bacteria such as Xa and Xap, the sensitivity to XOO3 subtype is high, but the kit is not suitable for XOO2 subtype, and the kit has the advantages of rapidness, sensitivity, specificity and low equipment dependence degree, and can be used for detecting the related bacteria of XOO3 subtype and XOO2 subtype. The method is suitable for non-diagnostic detection of field environments, port environments and the like.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically, it relates to a rapid detection method for rice bacterial blight pathogen based on RPA-CRISPR / Cas12b technology. Background Technology

[0002] Rice is a major food crop globally, and its production security is of paramount importance. Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (XOO), is a serious bacterial disease that severely damages rice production, leading to significant yield reductions or even crop failure. Therefore, developing efficient and accurate pathogen detection technologies is of great significance for the early control of this disease.

[0003] Currently, detection mainly relies on molecular detection techniques, such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). PCR technology has high sensitivity and specificity, but it is heavily dependent on thermal cycling equipment, making it cumbersome and time-consuming, and difficult to apply to rapid on-site detection. Isothermal amplification techniques such as LAMP and RPA overcome the dependence on temperature cycling to some extent, significantly shortening the amplification time and providing possibilities for the development of on-site detection.

[0004] However, these methods still have obvious limitations: First, most tests still require opening the lid for electrophoresis or test strip testing after completion, which increases the risk of aerosol contamination and can easily lead to false positive results; Second, the entire process involves multiple steps and is difficult to integrate, which limits its effectiveness in field environments such as ports and fields.

[0005] Therefore, there is an urgent need in this field for a novel detection method that combines high sensitivity and specificity, is easy to operate, is rapid, and does not require complex instruments, in order to achieve efficient on-site detection of rice bacterial blight pathogen. Summary of the Invention

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution: This invention provides a reagent combination for detecting rice bacterial blight pathogen (Xanthomonas oryzae pv. oryzae, XOO), including a specific RPA primer pair and sgRNA; The RPA primer pair consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; the sgRNA can target a specific sequence in the product amplified by the RPA primer pair.

[0007] Furthermore, the nucleotide sequence of the sgRNA is shown in SEQ ID NO.3.

[0008] This invention also provides a kit for detecting rice bacterial blight pathogen, comprising: (1) The reagent combination; (2) Cas12b protein; (3) ssDNA reporter molecule, wherein one end of the reporter molecule is labeled with a fluorescent reporter group and the other end is labeled with a quencher group.

[0009] This invention also provides a method for detecting rice bacterial blight pathogens for non-diagnostic purposes, comprising the following steps: S1. Using the nucleic acid of the sample to be tested as a template, perform RPA reaction using specific primer pairs with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; S2, CRISPR detection reaction: The amplification product is mixed with sgRNA, Cas12b protein and single-stranded DNA fluorescent reporter molecule with the sequence shown in SEQ ID NO.3 and incubated at an incubator. S3. Detect the fluorescence signal and determine whether the sample to be tested contains rice bacterial blight pathogen based on the fluorescence signal.

[0010] Furthermore, the RPA reaction is carried out at 43°C for 15 minutes; the isothermal incubation is carried out at 43°C for 10 minutes.

[0011] Furthermore, the RPA reaction, CRISPR detection reaction, and fluorescence signal detection are all automatically completed on the centrifugal microfluidic chip; the centrifugal microfluidic chip includes mutually isolated RPA reaction chambers and CRISPR detection chambers, wherein the RPA reaction chamber is pre-loaded with the RPA primer pair, and the CRISPR detection chamber is pre-loaded with the sgRNA. Beneficial effects

[0012] (1) This invention achieves ultra-high sensitivity detection of XOO pathogens (minimum 0.156 pg / reaction, approximately 2.2 copies / reaction) through carefully designed sgRNA and RPA primer pairs, with sensitivity comparable to qPCR. Furthermore, this system exhibits no cross-reactivity with five common closely related pathogens (Xa, Xap, Xam, Xav, and Xcm), demonstrating high specificity and effectively avoiding false positives, resulting in accurate and reliable detection. In addition, this detection system shows high sensitivity for the XOO3 subtype but is not applicable to the XOO2 subtype, further demonstrating the method's good subtype specificity.

[0013] (2) The entire detection process of the present invention, including RPA isothermal amplification (43℃, 15min) and CRISPR detection reaction (43℃, 10min), can be completed within 30 minutes, which greatly shortens the detection time and meets the needs of point-of-care testing (POCT).

[0014] (3) The present invention integrates the two steps of RPA amplification and CRISPR detection into a closed centrifugal microfluidic chip, realizing the whole process of "sample in, result out" closed tube operation, avoiding the risk of aerosol contamination caused by opening the cap to detect products in conventional methods, and significantly improving the reliability and accuracy of detection results.

[0015] (4) This invention simplifies complex multi-step operations into a single sample addition through reagent pre-freeze drying and chip-based design. The detection process is automatically completed by portable equipment, eliminating the need for complex thermal cyclers and professional operators, greatly reducing the barrier to entry and making it suitable for resource-limited environments such as grassroots units, ports, and field sites. Attached Figure Description

[0016] Figure 1 The following is a graph showing the performance screening results of three candidate sgRNAs targeting XOO in this embodiment of the invention: (A) Fluorescence curve; (B) Fluorescence change rate.

[0017] Figure 2 The figure shows the performance screening results of different RPA primer combinations for the XOO target in the embodiments of the present invention; (A) is the fluorescence curve; (B) is the fluorescence change rate.

[0018] Figure 3 The graph shows the sensitivity verification results of the XOO target in the detection method described in this invention; (A) is the fluorescence curve; (B) is the fluorescence change rate.

[0019] Figure 4 The image shows the results of the specificity verification of the XOO target by the detection method described in this invention; (A) is the fluorescence curve; (B) is the fluorescence change rate.

[0020] Figure 5 The diagram shows the compatibility verification results of the detection method for the XOO target described in this invention; (A) is the fluorescence curve; (B) is the fluorescence change rate.

[0021] Figure 6 The figure shows the results of the interference verification of the XOO target genome testing rapid extraction process of the detection method described in this invention; (A) is the fluorescence curve; (B) is the fluorescence change rate.

[0022] Figure 7 The following diagrams show the detection results of the rapid extraction process of XOO target bacterial solution at different concentrations according to the detection method of the present invention: (A) is the fluorescence curve; (B) is the fluorescence change rate.

[0023] Figure 8 The results of the bacterial lysis test of the XOO target non-rapid extraction system described in this invention are shown in Figure 1; (A) is the fluorescence curve; (B) is the fluorescence change rate.

[0024] Figure 9 The following is a graph showing the sensitivity verification results of the rapid extraction of XOO target bacterial solution using the detection method described in this invention; (A) is the fluorescence curve; (B) is the fluorescence change rate.

[0025] Figure 10 This is a diagram showing the initial detection effect of the chip for the XOO target nucleic acid by the detection method described in this invention.

[0026] Figure 11 This is a diagram illustrating the detection effect of the XOO target confirmed by the chip in the detection method described in this invention.

[0027] Figure 12 This is a graph showing the sensitivity verification results of the XOO target genome chip for the detection method described in this invention. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0029] The nucleotide sequence involved in this invention is shown below: SEQ ID NO.1: The sequence shown is the nucleotide sequence of RPA primer XOO-RPA-F3.

[0030] SEQ ID NO.2: The sequence shown is the nucleotide sequence of RPA primer XOO-RPA-R1.

[0031] SEQ ID NO.3: The sequence shown is the nucleotide sequence of the sgRNA target XOO-sgRNA.

[0032] SEQ ID NO.4: The sequence shown is the nucleotide sequence of the sgRNA target XOO-tale-2.

[0033] SEQ ID NO.5: The sequence shown is the nucleotide sequence of the sgRNA target XOO-rhs-1.

[0034] SEQ ID NO.6: The sequence shown is the nucleotide sequence of PCR primer Xoo-F.

[0035] SEQ ID NO.7: The sequence shown is the nucleotide sequence of PCR primer Xoo-R.

[0036] SEQ ID NO.8: The sequence shown is the nucleotide sequence of the PCR primer Xoo_rhsF1.

[0037] SEQ ID NO.9: The sequence shown is the nucleotide sequence of the PCR primer Xoo_rhsR1.

[0038] SEQ ID NO.10: The sequence shown is the nucleotide sequence of the PCR primer Xoo_avrF1.

[0039] SEQ ID NO.11: The sequence shown is the nucleotide sequence of the PCR primer Xoo_avrR1.

[0040] SEQ ID NO.12: The sequence shown is the nucleotide sequence of RPA primer XOO-RPA-F1.

[0041] SEQ ID NO.13: The sequence shown is the nucleotide sequence of RPA primer XOO-RPA-F2.

[0042] SEQ ID NO.14: The sequence shown is the nucleotide sequence of RPA primer XOO-RPA-R2. Example

[0043] 1. Experimental materials 1.1 Instruments and Equipment SynSor Portable Multi-Target Rapid Detection Device, XS-D-001 – Xunshi Biotechnology; QPCR Instrument - SLAN-96P – Shanghai Hongshi Medical Technology Co., Ltd.; PCR Instrument (TC-S / 96 / G / H(b)BA) – Hangzhou Bori Technology Co., Ltd.; Qubit 3.0 Fluorometer – Thermo Fisher Scientific; Nanodrop 2000 Ultra-Micro Spectrophotometer – Thermo Fisher Scientific; Metal Bath (DH100-2) – Hangzhou Ruicheng Instrument Co., Ltd.

[0044] 1.2 Reagents SynSor AaCas 12b (C2c1) (XS-R-002) – Xunshi Biosciences; SynSor DNA / RNA Isothermal Rapid Amplification Reagent (XS-R-101) – Xunshi Biosciences; SynSor CRISPR ssDNA Reporter (12b-FAM) (XS-R-201) – Xunshi Biosciences; SynSor sgRNA (XS-R-301) – Xunshi Biosciences; Gold MIX (Green) (TSE101) – Beijing Qingke Biotechnology Co., Ltd.; All water used is UltraPure™ Distilled Water, Dnase, Rnase, Free (10977-015) – Invitrogen (Shanghai) Trading Co., Ltd.

[0045] 2. Experimental Methods 2.1 Detection Target Targets were designed and developed for the target areas shown in Table 1.

[0046]

[0047] Eight nucleic acid samples and one bacterial culture sample were used to verify the actual sample performance. In addition, bacterial cultures for each target were tested. The corresponding sample information is shown in Table 2 below.

[0048]

[0049] 2.2 sgRNA Design and Validation 2.2.1 sgRNA Design Based on the selected region or commonly used target regions in the literature, and combined with the host background genome to be avoided, we use bioinformatics algorithms to score sgRNA designs based on factors such as PAM position, GC%, internal dimer structure, fragment structural openness, base position preference, and specificity. The score can be understood as a success probability; generally, three candidate fragments with scores above 40 are retained. If the scores are generally below 40, the number of candidate fragments needs to be increased to improve the success probability. Sequences in the spacer that pose a risk of disrupting the backbone secondary structure are directly excluded from the candidate list to avoid risks to specificity and sensitivity.

[0050] Based on the above design principles, sgRNAs were designed for each selected region, followed by synthesis and validation. The spacer sequences of the designed sgRNAs were analyzed for coverage and specificity using NCBI primer-BLAST. The alignment results showed that the designed sgRNA spacer sequences achieved 100% coverage within the tested species, indicating good inclusiveness; the number of bases matching across species was <15 nt, demonstrating good specificity. The sgRNAs used are shown in Table 3.

[0051]

[0052] 2.2.2 sgRNA Performance Verification 2.2.2.1 PCR Primer Design Based on the designed sgRNA location, PCR primers were designed within 100 bp upstream and downstream of the sgRNA (see Table 4). Primers were designed using commonly used PCR primer design software, with primer lengths controlled between 20-35 bp. If multiple sgRNA design sites exist within the same target fragment, the PCR amplification product (fragment length controlled within 500 bp) will contain all sgRNA binding sites, allowing all sgRNAs to be validated using the same template.

[0053]

[0054] 2.2.2.2 Template amplification verification PCR amplification was performed using plasmid / template DNA and corresponding primers to obtain high-concentration PCR products. Specific amplification systems are shown in Table 5, and the corresponding PCR reaction procedures are shown in Table 6.

[0055]

[0056]

[0057] The concentration of the amplification products of the target and the blank control was detected by Qubit double-stranded DNA nucleic acid fluorescent dye. The concentration of the target should be higher than 2 ng / μL and be clearly distinguishable from the concentration of the blank control.

[0058] 2.2.2.3 sgRNA Validation Prepare the CRISPR system according to the system in Table 7.

[0059]

[0060] Mix 1 μL (approximately 10–100 ng) of the PCR amplification product with the CRISPR system described above, and incubate at 43°C for 15 min using a qPCR instrument, collecting FAM fluorescence signals every minute. Based on the changes in the fluorescence signal curve, perform preliminary verification of the sgRNA performance or specificity. The endpoint where the fluorescence value curves of the target amplification product and the negative control stop rising should be significantly different.

[0061] The formula for calculating the fluorescence intensity growth rate is as follows: .

[0062] Here, Fluorescence Slope represents the fluorescence growth rate, Rn represents the fluorescence signal at minute n, and R1 represents the fluorescence signal at minute 1. A Fluorescence Slope greater than 0.5 is considered positive, and the Fluorescence Slope in the negative group must not exceed 0.2.

[0063] 2.3 Primer Design and Validation 2.3.1 RPA Primer Design Within the target detection range, fragments of 30-35 bases in length were selected as candidate RPA primers. To ensure stability and specificity, the GC content of RPA primers should be between 40% and 60%, and the RPA amplification fragment is typically 100-200 bp. Based on this primer design principle, this project designed three upstream and three downstream primers for RPA primer validation, and used NCBI Primer-BLAST to verify amplification coverage and specificity.

[0064] Based on the above design principles, RPA primers as shown in Table 8 were designed for each plasmid / template for subsequent synthesis and verification experiments.

[0065]

[0066] 2.4 Chip Verification 2.4.1 Chip Testing Process This section was validated using nucleic acids and bacterial cultures.

[0067] Using the system identified in section 3.2, the target nucleic acid samples were validated. First, the samples were rapidly extracted. Then, the sample lysis buffer was diluted 8-fold to prepare the RPA system. After thorough mixing, the samples were loaded onto the chip for detection. The positive or negative result was determined based on the captured fluorescence signal.

[0068] The chip testing process is shown in Table 9, with a total testing time of approximately 28 minutes.

[0069]

[0070] 3. Experimental Results 3.1 sgRNA screening results The fluorescence intensity of the CRISPR reaction was measured for each sgRNA and its corresponding template amplification product. The amplification curves and fluorescence growth rate analysis results are shown below. Figure 1 The results of fluorescence value changes are shown in Table 10. Analysis results show that the XOO target XOO-sgRNA showed good efficacy.

[0071]

[0072] 3.2 RPA primer screening results The nucleic acid sample was diluted to 10 ng / μL to screen different combinations of RPA primers. A two-step CRISPR trans reaction was used. The first step was to perform RPA amplification, and then the RPA amplification product was mixed with the CRISPR reaction solution to perform the CRISPR reaction.

[0073] For the target amplification product, a change in fluorescence slope exceeding 0.5 within 15 minutes is considered a valid detection; for the negative control and blank control, the change in original fluorescence value within 15 minutes should be less than 0.2.

[0074] The effective amplification rates of each primer at each dilution gradient are shown in Table 11.

[0075]

[0076] The analysis results show that: Most of the primers designed for the XOO target were able to amplify the sample. After comparing their performance, the RPA-XOO-F3R1 primer pair, which had better amplification performance, was selected. Figure 2 Proceed to the next step of sensitivity verification.

[0077] 3.3 Construction and Validation of the Testing System 3.3.1 Validation of the sensitivity of the detection system A serial dilution was performed using the genomic template, with the genomic addition levels per unit reaction set at 156 pg, 15.6 pg, 7.8 pg, 1.56 pg, 0.78 pg, 0.156 pg, and 0.078 pg, adjusted appropriately based on the effects on different targets. The primer and sgRNA combinations selected in section 3.2, along with the same CRISPR system, were used to validate the sensitivity of the target. Each dilution was repeated three times for validation.

[0078] The amplification curves and fluorescence signal growth rates for sensitivity verification of each target are shown below, XOO ( Figure 3 The minimum detectable amount is 0.156 pg of nucleic acid sample.

[0079] Based on the significance analysis of the sensitivity verification experiment results, the detection limits of the primers and corresponding targets can be preliminarily determined as shown in Table 12.

[0080]

[0081] Based on previous studies on the relationship between genome copy number and genome quality, the XOO sensitivity is approximately 2.2 copies / T.

[0082] 3.3.2 Validation of the Specificity of the Detection System Five cross-reactions were used for target specificity testing, with the target species serving as a positive control, the cross-reactions as a specificity validation test group, and water as a negative control template. The target specificity was validated using the primer and sgRNA combinations selected in section 3.2, along with the same CRISPR system. Each cross-reaction was performed twice for verification.

[0083] The results of the XOO target specificity verification are shown in the figure. Figure 4 The validation results showed that the XOO target was positive in XOO samples, but negative in the five cross-species (Xa, Xap, Xam, Xav, and Xcm), indicating that the XOO target has good specificity in cross-species.

[0084] 3.3.3 Sample compatibility testing of the testing system The nucleic acid was diluted to 10 ng / μL for this part of the verification.

[0085] Utilizing existing detection systems, the target nucleic acid was validated using actual samples. Each actual sample was tested twice for repeated validation.

[0086] The amplification curves and fluorescence signal growth rates of different subtype genomic samples are shown below: The XOO target is compatible with XOO3, but not with XOO2 (see below). Figure 5 .

[0087] 3.4 Development of a rapid extraction process for bacterial culture samples 3.4.1 Evaluation of interference from rapid extraction process on the system The rapid extraction procedure using the target genome assay was used to test for interference with the detection system. The original sample mixture (hereinafter referred to as the stock solution) was used as a positive control, and the samples prepared using the rapid extraction procedure were used as the test group. Validation was performed using the primer and sgRNA combinations selected in section 3.2, along with the same CRISPR system. Figure 6 Each cross-reaction was verified by repeating the experiment twice.

[0088] 3.4.2 Feasibility Verification of the Rapid Extraction System for Bacterial Fluid The bacterial suspension was diluted 10 times and 100 times for this part of the verification.

[0089] The target bacterial culture samples were validated using the existing detection system. Each actual sample was tested twice for repeated validation.

[0090] The amplification curves and fluorescence signal growth rates of bacterial suspension samples at different concentrations are shown below. XOO bacterial suspension was undetectable at both concentrations. Figure 7 Overall, the rapid extraction process allows for the lysis of bacterial cultures for detection.

[0091] 3.4.3 Performance Verification of Bacterial Fluid in Non-Rapid Extraction System The bacterial culture sample was tested at 1E6 CFU / mL. The primer, template, and sgRNA combinations selected in section 3.2, along with the same CRISPR system, were used. The rapid extraction system was used as a positive control, and the non-rapid extraction system (physiological saline) was tested for its lysis effect on the bacterial culture. Each test was performed twice for verification.

[0092] The results of the saline lysate bacterial suspension detection are shown below. No positive signals were observed in either the rapid extraction of the XOO target bacterial suspension or the saline lysate, consistent with 3.4.2, indicating subtype inclusivity. See details below. Figure 8 .

[0093] 3.4.4 Sensitivity Validation of Rapid Bacterial Fluid Extraction Procedure The bacterial culture samples were serially diluted with gradients including 1E6 CFU / mL, 1E5 CFU / mL, 1E4 CFU / mL, 1E3 CFU / mL, 1E2 CFU / mL, 1E1 CFU / mL, and 1 CFU / mL, corresponding to single-reaction colony counts of 156 CFU, 15.6 CFU, 1.56 CFU, 0.156 CFU, 0.0156 CFU, 0.00156 CFU, and 0.000156 CFU, respectively. These values ​​were adjusted appropriately based on the effectiveness against different targets. The primer, template, and sgRNA combinations selected in section 3.2, along with the same CRISPR system, were used to validate the sensitivity of the target bacterial culture. Each gradient was validated in duplicate.

[0094] The amplification curves and fluorescence signal growth rates for sensitivity validation of the bacterial culture are shown below: XOO target bacterial culture samples were not detected ( Figure 9 ).

[0095] 3.5 Chip Testing Results 3.5.1 Initial chip test results The nucleic acid sample was diluted to a concentration of 10 ng / μL. Initial testing was performed on a chip using a rapid extraction procedure, and the target validation results are as follows: Figure 10XOO2 was not detected, while XOO3 was detectable.

[0096] 3.5.2 Chip Confirmation of XOO Target Detection Effectiveness The XOO target nucleic acid sample was diluted to 10 ng / μL and validated using the target primers and sgRNA screened in section 3.2. A two-step CRISPR reverse reaction was employed. The first step involved RPA amplification, followed by mixing the RPA amplification product with the CRISPR reaction solution for the CRISPR reaction. After completing the chip fabrication process, the results were interpreted based on the brightness changes of the detection wells. In the chip setup, well 3 corresponds to the XOO target.

[0097] Target validation results are as follows Figure 11 The XOO target detection system corresponds to well 3 on the chip; when the sample is the XOO genome, it is positive.

[0098] 3.5.3 Sensitivity of Rapid Extraction and Detection of Three-Target Bacterial Fluids under Chip Process The bacterial culture or genomic sample was serially diluted, and the target primers and sgRNA selected in section 3.2 were used for verification. A two-step CRISPR trans reaction was used. The first step was to perform RPA amplification, and then the RPA amplification product was mixed with the CRISPR reaction solution for CRISPR reaction. The positive and negative results were determined using the built-in algorithm of the instrument (wells with square boxes around them are positive wells).

[0099] XOO target genome chip detection results are as follows: Figure 12 The XOO target chip can detect genomic samples, and in the chip process, a minimum of 1 pg / T of genomic sample can be detected per well, which is approximately equivalent to 22 copies per reaction.

[0100] In summary, this invention develops an isothermal amplification detection method based on RPA and CRISPR for the XOO target, eliminating the dependence on complex instrumentation platforms required by traditional molecular biology methods and greatly simplifying the detection platform. Through system establishment and validation, the overall detection time for the target has been reduced to less than half an hour, with further potential for reduction in the future. Simultaneously, the sensitivity, specificity, and POCT platform validation of the target detection technology were conducted. The results show that the developed detection method has good sensitivity and specificity, and the actual sample validation results are consistent with the gold standard detection method. Detailed detection limits and validation data for the target are as follows: XOO-sgRNA, used in conjunction with RPA-XOO-F3 / R1, showed good detection results for the XOO target sgRNA, achieving a minimum detection rate of 0.156 pg per reaction, equivalent to approximately 2.2 copies per reaction. It is specific to the five cross-species groups (Xa, Xap, Xam, Xav, and Xcm), but does not include the XOO2 subtype, although it does include the XOO3 subtype. A rapid extraction process on the chip can detect genome samples as low as 1 pg / T, equivalent to approximately 14.1 copies per reaction.

Claims

1. A reagent combination for detecting Xanthomonas oryzae pv. oryzae (XOO), the pathogen of rice bacterial blight, characterized in that, Including specific RPA primer pairs and sgRNA; The RPA primer pair consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; the sgRNA can target a specific sequence in the product amplified by the RPA primer pair.

2. The reagent combination according to claim 1, characterized in that, The nucleotide sequence of the sgRNA is shown in SEQ ID NO.

3.

3. A reagent kit for detecting rice bacterial blight pathogen, characterized in that, Include: (1) The reagent combination according to claim 1 or 2; (2) Cas12b protein; (3) ssDNA reporter molecule, wherein one end of the reporter molecule is labeled with a fluorescent reporter group and the other end is labeled with a quencher group.

4. A method for detecting rice bacterial blight pathogens (not for diagnostic purposes), characterized in that, Includes the following steps: S1. Using the nucleic acid of the sample to be tested as a template, perform RPA reaction using specific primer pairs with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; S2, CRISPR detection reaction: The amplification product is mixed with sgRNA, Cas12b protein and single-stranded DNA fluorescent reporter molecule with the sequence shown in SEQ ID NO.3 and incubated at an incubator. S3. Detect the fluorescence signal and determine whether the sample to be tested contains rice bacterial blight pathogen based on the fluorescence signal.

5. The detection method according to claim 4, characterized in that, The RPA reaction was carried out at 43°C for 15 minutes; the isothermal incubation was carried out at 43°C for 10 minutes.

6. The detection method according to claim 4, characterized in that, The RPA reaction, CRISPR detection reaction, and fluorescence signal detection are all automatically completed on a centrifugal microfluidic chip; the centrifugal microfluidic chip includes an RPA reaction chamber and a CRISPR detection chamber that are isolated from each other, wherein the RPA reaction chamber is pre-loaded with the RPA primer pair of claim 1, and the CRISPR detection chamber is pre-loaded with the sgRNA of claim 2.

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