A rapid detection method for rice false smut fungus based on RPA-CRISPR / Cas12b technology

Abstract

This invention discloses a rapid detection method for rice blight pathogens based on RPA-CRISPR / Cas12b technology, belonging to the field of nucleic acid detection technology. The reagent combination includes specific RPA primer pairs (SEQ ID NO.1 and SEQ ID NO.2) and sgRNA (SEQ ID NO.3). The method combines recombinase polymerase amplification (RPA) with CRISPR / Cas12b technology. RPA isothermally amplifies the target nucleic acid, and the Cas12b / sgRNA complex recognizes the amplified product and activates its trans-cleavage activity, thereby cleaving a fluorescent reporter molecule to generate a signal. This invention integrates an optimized reaction system into a centrifugal microfluidic chip, achieving fully automated closed-loop detection with "sample in, result out." The method can be completed within 30 minutes, with a sensitivity of up to 0.156 pg / reaction, high specificity, and is suitable for rapid detection in fields, ports, and other on-site locations.

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 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. Bacterial rice blight, caused by Burkholderia glumae (BG), 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 prevention and 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 blight pathogens. Summary of the Invention

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0007] This invention provides a reagent combination for detecting Burkholderia glumae, the causal agent of rice wilt, comprising a specific RPA primer pair and sgRNA;

[0008] 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 is capable of targeting a specific sequence in the product amplified by the RPA primer pair, and its nucleotide sequence is shown in SEQ ID NO.3.

[0009] The present invention also provides a kit for detecting rice blight pathogens, comprising:

[0010] (1) The reagent combination described above;

[0011] (2) Cas12b protein;

[0012] (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.

[0013] This invention also provides a method for detecting rice blight pathogens for non-diagnostic purposes, comprising the following steps:

[0014] S1. Using the nucleic acid of the sample to be tested as a template, perform an RPA reaction using the specific RPA primer pair in the reagent combination;

[0015] S2, CRISPR detection reaction: The amplification product is mixed with the sgRNA, Cas12b protein and single-stranded DNA fluorescent reporter molecule in the reagent combination and incubated at a constant temperature.

[0016] S3. Detect the fluorescence signal and determine whether the sample to be tested contains rice blight pathogen based on the fluorescence signal.

[0017] 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.

[0018] Furthermore, the RPA reaction, CRISPR detection reaction, and fluorescence signal detection are all automatically completed on a centrifugal microfluidic chip; the centrifugal microfluidic chip includes mutually isolated RPA reaction chambers and CRISPR detection chambers pre-loaded with the RPA primer pairs and sgRNA from the reagent combination. Beneficial effects

[0019] (1) This invention achieves ultra-high sensitivity detection of BG pathogens through carefully designed sgRNA and RPA primer pairs (the limit of detection for rice wilt pathogen genomic DNA is 0.156 pg / reaction, approximately 5.7 copies / reaction; the limit of detection for bacterial suspension is 15.6 CFU / reaction), with sensitivity comparable to qPCR. This system exhibits no cross-reactivity with five common closely related pathogens, demonstrating high specificity and effectively avoiding false positives, resulting in accurate and reliable detection. Furthermore, this detection system shows high sensitivity to BG2 and BG3 subtypes, further demonstrating the good subtype specificity of this method.

[0020] (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).

[0021] (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.

[0022] (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

[0023] Figure 1 The following is a graph showing the performance screening results of three candidate sgRNAs for the BG target in this embodiment of the invention: (A) Fluorescence curve; (B) Fluorescence change rate.

[0024] Figure 2 The following figures show the performance screening results of different RPA primer combinations for the BG target in this embodiment of the invention: (A) Fluorescence curve; (B) Fluorescence change rate.

[0025] Figure 3 The following figures show the sensitivity verification results of the detection method for the BG target described in this invention: (A) Fluorescence curve; (B) Fluorescence change rate.

[0026] Figure 4 The following figures show the results of the specificity verification of the BG target in the detection method described in this invention: (A) Fluorescence curve; (B) Fluorescence change rate.

[0027] Figure 5 The following figures show the results of the BG target subtype compatibility verification of the detection method described in this invention: (A) Fluorescence curve; (B) Fluorescence change rate.

[0028] Figure 6 Figure 1 shows the results of the rapid extraction process for BG target genome testing of the detection method described in this invention and its interference verification on the system; (A) fluorescence curve; (B) fluorescence change rate.

[0029] Figure 7 The following are the detection results of the rapid extraction process of BG target bacterial solution at different concentrations according to the detection method of the present invention: (A) Fluorescence curve; (B) Fluorescence change rate.

[0030] Figure 8 The results of the bacterial lysis test of the non-rapid extraction system of BG target described in the detection method of the present invention are shown in the figure; (A) fluorescence curve; (B) fluorescence change rate.

[0031] Figure 9 The graph shows the sensitivity verification results of the rapid extraction process of BG target bacterial solution in the detection method of the present invention; (A) is the fluorescence curve; (B) is the fluorescence change rate.

[0032] Figure 10 This is a diagram showing the initial detection effect of the chip for BG target nucleic acid by the detection method described in this invention.

[0033] Figure 11 The image shows the effect of different primer pairs on the BG target tested by the detection method chip described in this invention.

[0034] Figure 12 This is a diagram showing the detection effect of the chip in the detection method described in this invention on the BG target.

[0035] Figure 13 This is a graph showing the sensitivity verification results of the BG target bacterial culture chip for the detection method described in this invention. Detailed Implementation

[0036] 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.

[0037] The nucleotide sequence involved in this invention is shown below:

[0038] SEQ ID NO.1: The sequence shown is the nucleotide sequence of RPA primer RPA-BG-F3.

[0039] SEQ ID NO.2: The sequence shown is the nucleotide sequence of RPA primer RPA-BG-R4.

[0040] SEQ ID NO.3: The sequence shown is the nucleotide sequence of the sgRNA target Bg-sgRNA.

[0041] SEQ ID NO.4: The sequence shown is the nucleotide sequence of the sgRNA target Bg-ITS-2.

[0042] SEQ ID NO.5: The sequence shown is the nucleotide sequence of the sgRNA target Bg-GyrB-1.

[0043] SEQ ID NO.6: The sequence shown is the nucleotide sequence of PCR primer Bg-F.

[0044] SEQ ID NO.7: The sequence shown is the nucleotide sequence of PCR primer Bg-R.

[0045] SEQ ID NO.8: The sequence shown is the nucleotide sequence of PCR primer Bkg_GB_gyrBF1.

[0046] SEQ ID NO.9: The sequence shown is the nucleotide sequence of PCR primer Bkg_GB_gyrBR1.

[0047] SEQ ID NO.10: The sequence shown is the nucleotide sequence of PCR primer Bg-impEF1.

[0048] SEQ ID NO.11: The sequence shown is the nucleotide sequence of PCR primer Bg-impER1.

[0049] SEQ ID NO.12: The sequence shown is the nucleotide sequence of RPA primer RPA-BG-F1.

[0050] SEQ ID NO.13: The sequence shown is the nucleotide sequence of RPA primer RPA-BG-F2.

[0051] SEQ ID NO.14: The sequence shown is the nucleotide sequence of RPA primer RPA-BG-R1.

[0052] SEQ ID NO.15: The sequence shown is the nucleotide sequence of RPA primer RPA-BG-R2.

[0053] SEQ ID NO.16: The sequence shown is the nucleotide sequence of RPA primer RPA-BG-R3. Example

[0054] 1. Experimental materials

[0055] 1.1 Instruments and Equipment

[0056] 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.

[0057] 1.2 Reagents

[0058] 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.

[0059] 2. Experimental Methods

[0060] 2.1 Detection Target

[0061] Targets were designed and developed for the target areas shown in Table 1.

[0062]

[0063] Eight nucleic acid samples and one bacterial culture sample were used to verify the actual sample performance. In addition, bacterial culture was also tested. The corresponding sample information is shown in Table 2 below.

[0064]

[0065] 2.2 sgRNA Design and Validation

[0066] 2.2.1 sgRNA Design

[0067] 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.

[0068] 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.

[0069]

[0070] 2.2.2 sgRNA Performance Verification

[0071] 2.2.2.1 PCR Primer Design

[0072] 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.

[0073]

[0074] 2.2.2.2 Template amplification verification

[0075] 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.

[0076]

[0077]

[0078] 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.

[0079] 2.2.2.3 sgRNA Validation

[0080] Prepare the CRISPR system according to the system in Table 7.

[0081]

[0082] 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.

[0083] The formula for calculating the fluorescence intensity growth rate is as follows: .

[0084] 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.

[0085] 2.3 Primer Design and Validation

[0086] 2.3.1 RPA Primer Design

[0087] 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.

[0088] 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.

[0089]

[0090] 2.4 Chip Verification

[0091] 2.4.1 Chip Testing Process

[0092] This section was validated using nucleic acids and bacterial cultures.

[0093] 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.

[0094] The chip testing process is shown in Table 9, with a total testing time of approximately 28 minutes.

[0095]

[0096] 3. Experimental Results

[0097] 3.1 sgRNA screening results

[0098] 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 BG target Bg-sgRNA showed better efficacy.

[0099]

[0100] 3.2 RPA primer screening results

[0101] 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.

[0102] 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.

[0103] The effective amplification rates of each primer at each dilution gradient are shown in Table 11.

[0104] The analysis results show that:

[0105] All the primers designed for the BG target were able to amplify the sample. After comparing their performance, the RPA-BG-F3 / R4 primer pair with better amplification performance was selected. Figure 2 Proceed to the next step of sensitivity verification.

[0106]

[0107] 3.3 Construction and Validation of the Testing System

[0108] 3.3.1 Validation of the sensitivity of the detection system

[0109] 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.

[0110] The amplification curves and fluorescence signal growth rates for sensitivity verification of each target are shown below, BG ( Figure 3 Both can detect nucleic acid samples as low as 0.156 pg.

[0111] 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.

[0112]

[0113] Based on previous studies on the relationship between genome copy number and genome quality, the BG sensitivity is approximately 5.7 copies / T.

[0114] 3.3.2 Validation of the Specificity of the Detection System

[0115] 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.

[0116] The results of the specificity verification of the BG target are shown in the figure below. Figure 4 The validation results showed that the BG target was positive in BG samples, but negative in 5 cross-species, indicating that the BG target has good specificity in cross-species.

[0117] 3.3.3 Sample compatibility testing of the testing system

[0118] The nucleic acid was diluted to 10 ng / μL for this part of the verification.

[0119] Utilizing existing detection systems, the target nucleic acid was validated using actual samples. Each actual sample was tested twice for repeated validation.

[0120] The amplification curves and fluorescence signal growth rates of different subtype genomic samples are shown below. The BG target is compatible with BG2 and BG3 subtypes (see...). Figure 5 ).

[0121] 3.4 Development of a rapid extraction process for bacterial culture samples

[0122] 3.4.1 Evaluation of interference from rapid extraction process on the system

[0123] To test the interference of the rapid extraction procedure on 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. Each cross-reaction was repeated twice for verification.

[0124] BG target ( Figure 6 The verification results showed that the original solution tested positive, and the rapid extraction sample test results were similar to those of the original solution, with no significant decrease.

[0125] 3.4.2 Feasibility Verification of the Rapid Extraction System for Bacterial Fluid

[0126] The bacterial suspension was diluted 10 times and 100 times for this part of the verification.

[0127] The target bacterial culture samples were validated using the existing detection system. Each actual sample was tested twice for repeated validation.

[0128] The amplification curves and fluorescence signal growth rates of bacterial suspension samples at different concentrations are shown below. The BG target was detectable in both concentrations of bacterial suspension samples. Figure 7 Overall, the rapid extraction process allows for the lysis of bacterial cultures for testing.

[0129] 3.4.3 Performance Verification of Bacterial Fluid in Non-Rapid Extraction System

[0130] 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.

[0131] The detection results of physiological saline lysate bacterial culture are shown below, BG target ( Figure 8 No positive results could be detected by lysis with physiological saline, which is significantly different from the effect of the rapid extraction process, further demonstrating that the rapid extraction process has a significant effect on nucleic acid release from bacterial culture.

[0132] 3.4.4 Sensitivity Validation of Rapid Bacterial Fluid Extraction Procedure

[0133] 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.

[0134] The amplification curves and fluorescence signal growth rates for sensitivity verification of the bacterial culture are shown below. The lowest detectable level for the BG target is 15.6 CFU / T of bacterial culture samples. Figure 9 ).

[0135] 3.5 Chip Testing Results

[0136] 3.5.1 Initial chip test results

[0137] The nucleic acid sample was diluted to a concentration of 10 ng / μL. Initial testing was performed on the chip using a rapid extraction protocol, and the results were verified as follows: Figure 10 BG target genome was not detected.

[0138] 3.5.2 Chip testing of BG target primer pairs

[0139] Nucleic acid samples were diluted to 10 ng / μL, and four pairs of RPA primers with good performance selected in section 3.2—RPA-BG-F1 / R3, RPA-BG-F1 / R4, RPA-BG-F2 / R4, and RPA-BG-F3 / R4—were used for chip testing. A two-step CRISPR reaction was employed. The first step was to perform RPA amplification, followed by mixing the RPA amplification product with the CRISPR reaction solution for a CRISPR reaction. The positive and negative results were determined based on the captured fluorescence signals.

[0140] The validation results of different primer pairs for the BG target are as follows: Figure 11 RPA-BG-F1 / R3, RPA-BG-F2 / R4, and RPA-BG-F3 / R4 were not detected, while RPA-BG-F1 / R4 showed a positive signal.

[0141] 3.5.3 Chip confirmation of BG target detection effectiveness

[0142] BG target nucleic acid samples were diluted to 10 ng / μL and validated using the target primers and sgRNA screened in section 3.2. The BG primer pair was replaced with RPA-BG-F1 / R4 for chip testing, employing a two-step CRISPR reverse reaction. 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, results were interpreted based on changes in the brightness of the detection wells. In the chip setup, well 1 corresponds to the BG target.

[0143] The verification results are as follows Figure 12 The BG target detection system corresponds to well 1 on the chip, and it can be seen that it is positive only when the sample is a BG sample.

[0144] 3.5.4 Sensitivity of Rapid Extraction and Detection of Target Bacterial Fluids under Chip-Based Processing

[0145] 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).

[0146] BG target bacterial culture chip detection results are as follows: Figure 13 The BG target can detect bacterial culture after rapid extraction on the chip, with the entire process completed, and can detect bacterial culture samples as low as 15.6 CFU.

[0147] In summary, this invention develops an isothermal amplification detection method based on RPA and CRISPR for the BG target, eliminating the dependence on complex instrument 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 room for reduction in the future. Simultaneously, the sensitivity, specificity, and POCT platform of the target detection technology were validated. 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:

[0148] The BG target sgRNA was identified using Bg-sgRNA, which can be tested with primer RPA-BG-F3R4. The lowest detectable amount of genome per reaction is 0.156 pg, approximately equivalent to 5.7 copies per reaction, and it is specific to BG-CK1–BG-CK5. The rapid extraction procedure combined with the detection system can detect bacterial cultures as low as 15.6 CFU / T, and the lowest detectable amount of bacterial cultures on the microarray is 15.6 CFU / T.

Claims

1. A reagent combination for detecting Burkholderia glumae, characterized by, 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, and its nucleotide sequence is shown in SEQ ID NO.3; The reagent combination is used for detection in a centrifugal microfluidic chip, which includes mutually isolated RPA reaction chambers and CRISPR detection chambers. The RPA reaction chambers are pre-loaded with the RPA primer pair, and the CRISPR detection chambers are pre-loaded with the sgRNA.

2. A reagent kit for detecting rice blight pathogen, characterized in that, Include: (1) The reagent combination according to claim 1; (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.

3. A method for detecting rice 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 in the RPA reaction chamber of the centrifugal microfluidic chip using the specific RPA primer pair in the reagent combination of claim 1. S2, CRISPR detection reaction: The amplification product is transferred to the CRISPR detection chamber by centrifugation, mixed with pre-placed sgRNA, Cas12b protein and single-stranded DNA fluorescent reporter molecule and incubated at an incubator; the nucleotide sequence of the sgRNA is shown in SEQ ID NO.3; S3. Detect the fluorescence signal and determine whether the sample to be tested contains rice blight pathogen based on the fluorescence signal.

Images