RPA-CRISPR / Cas12b technology-based rapid detection method for rice blight germs

Abstract

The invention discloses a rapid detection method for rice blight germs based on an RPA-CRISPR / Cas12b technology, and belongs to the technical field of nucleic acid detection. The reagent combination comprises a specific RPA (recombinase polymerase amplification) primer pair (SEQ ID NO.1 and SEQ ID NO.2) and sgRNA (sgRNA) (SEQ ID NO.3). According to the method, recombinase polymerase amplification (RPA) and a CRISPR / Cas12b technology are combined, isothermal amplification is carried out on target nucleic acid through RPA, a Cas12b / sgRNA compound is used for recognizing an amplification product and activating the trans-cleavage activity of the amplification product, and therefore fluorescent reporter molecules are cleaved to generate signals. According to the invention, an optimized reaction system is integrated on the centrifugal micro-fluidic chip, so that full-flow closed-tube automatic detection of'sample feeding and result discharging 'is realized. The method can be completed within 30 minutes, the sensitivity is as high as 0.156 pg / reaction, the specificity is high, and the method is suitable for rapid detection in fields, ports and other fields.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nucleic acid detection, and particularly relates to a rapid detection method for rice bacterial seedling blight bacteria based on RPA-CRISPR / Cas12b technology. BACKGROUND

[0002] Rice is a major food crop in the world, and its production safety is crucial. Rice bacterial seedling blight caused by Burkholderia glumae (BG) is a serious bacterial disease that can cause significant yield reduction or even total loss, so developing an efficient and accurate pathogen detection technology is of great significance for early prevention and control of the 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 severely dependent on a thermal cycler, is complicated to operate, and takes a long time, making it difficult to apply to on-site rapid 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 the possibility for developing on-site detection.

[0004] However, these methods still have obvious limitations: first, most of them still need to be opened after detection for electrophoresis or test strip detection, increasing the risk of aerosol contamination and leading to false positive results; second, the entire process involves multiple steps of operation, which is difficult to integrate, limiting its application effectiveness in on-site environments such as ports and fields.

[0005] Therefore, there is an urgent need in the art for a new detection method that has high sensitivity and specificity, is easy to operate, is fast, and does not require complex instruments, in order to achieve on-site efficient detection of rice bacterial seedling blight bacteria. SUMMARY

[0006] To achieve the above application purposes, the application adopts the following technical solutions: The application provides a reagent combination for detecting rice bacterial seedling blight bacteria Burkholderia glumae, comprising a specific RPA primer pair and an 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 as shown in SEQ ID NO. 3.

[0007] The application also provides a kit for detecting rice bacterial seedling blight bacteria, comprising: (1) the reagent combination; (2) a Cas12b protein; (3) a ssDNA reporter molecule, which is labeled with a fluorescent reporter group at one end and a quencher group at the other end.

[0008] The application also provides a detection method for Magnaporthe oryzae for non-diagnostic purposes, comprising the following steps: S1, using the nucleic acid of a to-be-tested sample as a template, performing RPA reaction using specific RPA primer pairs in the reagent combination; S2, CRISPR detection reaction: mixing the amplification product with sgRNA, Cas12b protein and single-stranded DNA fluorescent reporter molecule in the reagent combination and performing constant-temperature incubation; S3, detecting the fluorescent signal, and determining whether the to-be-tested sample contains Magnaporthe oryzae according to the fluorescent signal.

[0009] Further, the RPA reaction is performed at 43℃ for 15 minutes; and the constant-temperature incubation is performed at 43℃ for 10 minutes.

[0010] Further, the RPA reaction, CRISPR detection reaction and fluorescent signal detection are all automatically completed on a centrifugal microfluidic chip; the centrifugal microfluidic chip comprises an RPA reaction cavity and a CRISPR detection cavity, which are mutually isolated and in which the RPA primer pairs and sgRNA in the reagent combination are pre-disposed. Advantages

[0011] (1) The application realizes ultra-high sensitivity detection of BG pathogenic bacteria (the minimum detection limit for Magnaporthe oryzae genomic DNA is 0.156 pg / reaction, about 5.7 copies / reaction; and the minimum detection limit for bacterial solution is 15.6 CFU / reaction) through the carefully designed sgRNA and RPA primer pairs, and the sensitivity is comparable to that of qPCR method. The system has no cross-reaction with five common close pathogenic bacteria, has strong specificity, effectively avoids false positive results, and the detection result is accurate and reliable. In addition, the detection system has high sensitivity to BG2 and BG3 subtypes, which also proves that the method has good subtype specificity.

[0012] (2) The entire detection process of the application, including RPA constant-temperature amplification (43℃, 15min) and CRISPR detection reaction (43℃, 10min), can be completed within 30 minutes, greatly shortening the detection time and meeting the needs of on-site rapid detection (POCT).

[0013] (3) The RPA amplification and CRISPR detection two steps are integrated in the closed centrifugal microfluidic chip in the application, realizes the whole process closed tube operation of "sample in, result out", avoids the aerosol pollution risk caused by opening cover to detect product in the conventional method, and significantly improves the reliability and accuracy of the detection result.

[0014] (4) The application simplifies the complex multi-step operation into one-time sample adding through reagent pre-freeze drying and chip design, and the detection process is automatically completed by relying on a portable device, without the need of complex thermal cycler and professional operators, so that the use threshold is greatly reduced, and the application is suitable for limited resource environments such as grassroots units, ports and fields. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a performance screening result graph of three candidate sgRNAs of BG target in the embodiment of the application; (A) fluorescence curve; (B) fluorescence change rate.

[0016] Figure 2 It is a performance screening result graph of different RPA primer combinations of BG target in the embodiment of the application; (A) fluorescence curve; (B) fluorescence change rate.

[0017] Figure 3 It is a sensitivity verification result graph of the BG target of the detection method; (A) fluorescence curve; (B) fluorescence change rate.

[0018] Figure 4 It is a specificity verification confirmation result graph of the BG target of the detection method; (A) fluorescence curve; (B) fluorescence change rate.

[0019] Figure 5 It is a subtype compatibility verification result graph of the BG target of the detection method; (A) fluorescence curve; (B) fluorescence change rate.

[0020] Figure 6 It is a result graph of interference verification of the BG target genome test rapid extraction process of the detection method; (A) fluorescence curve; (B) fluorescence change rate.

[0021] Figure 7 It is a detection effect graph of the BG target bacteria liquid of different concentrations of the rapid extraction process of the detection method; (A) fluorescence curve; (B) fluorescence change rate.

[0022] Figure 8 It is a bacteria liquid lysis test result graph of the BG target of the non-rapid extraction system of the detection method; (A) fluorescence curve; (B) fluorescence change rate.

[0023] Figure 9The figure is a sensitivity verification result of a BG target bacteria liquid rapid extraction process of the detection method.

[0024] Figure 10 The figure is a BG target nucleic acid effect of the detection method chip initial detection.

[0025] Figure 11 The figure is a BG target different primer pair effect of the detection method chip test.

[0026] Figure 12 The figure is a BG target detection effect of the detection method chip confirmation.

[0027] Figure 13 The figure is a BG target bacteria liquid chip sensitivity verification result of the detection method. DETAILED DESCRIPTION

[0028] In order to make the person skilled in the art better understand the technical solutions in the present application, the following will combine the embodiments to make the present application further detailed description.

[0029] The nucleotide sequence involved in the present application is as follows: SEQ ID NO. 1: the sequence shown is the nucleotide sequence of RPA primer RPA-BG-F3.

[0030] SEQ ID NO. 2: the sequence shown is the nucleotide sequence of RPA primer RPA-BG-R4.

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

[0032] SEQ ID NO. 4: the sequence shown is the nucleotide sequence of sgRNA target Bg-ITS-2.

[0033] SEQ ID NO. 5: the sequence shown is the nucleotide sequence of sgRNA target Bg-GyrB-1.

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

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

[0036] SEQ ID NO. 8: the sequence shown is the nucleotide sequence of PCR primer Bkg_GB_gyrBF1.

[0037] SEQ ID NO. 9: The sequence shown is the nucleotide sequence of PCR primer Bkg_GB_gyrBR1.

[0038] SEQ ID NO. 10: The sequence shown is the nucleotide sequence of PCR primer Bg-impEF1.

[0039] SEQ ID NO. 11: The sequence shown is the nucleotide sequence of PCR primer Bg-impER1.

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

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

[0042] SEQ ID NO. 14: The sequence shown is the nucleotide sequence of RPA primer RPA-BG-R1.

[0043] SEQ ID NO. 15: The sequence shown is the nucleotide sequence of RPA primer RPA-BG-R2.

[0044] SEQ ID NO. 16: The sequence shown is the nucleotide sequence of RPA primer RPA-BG-R3. Examples

[0045] 1. Experimental materials 1.1. Instruments and equipment SynSor portable multi-target rapid detection equipment, XS-D-001 - Xunxi Biology; QPCR instrument - SLAN-96P - Shanghai Hongshi Medical Technology Co., Ltd.; PCR instrument (TC-S / 96 / G / H(b)BA) - Hangzhou Boyi Technology Co., Ltd.; Qubit 3.0 fluorometer - Thermo Fisher Scientific; Nanodrop 2000 ultramicro spectrophotometer - Thermo Fisher Scientific; Metal bath (DH100-2) - Hangzhou Ruicheng Instrument Co., Ltd.

[0046] 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.

[0047] 2. Experimental Methods 2.1 Detection Target Targets were designed and developed for the target areas shown in Table 1.

[0048]

[0049] 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.

[0050]

[0051] 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.

[0052] Based on the above design principles, sgRNAs were designed for each selected region for subsequent synthesis and verification. The spacer sequence of the designed sgRNA was analyzed for coverage and specificity using NCBI primer-BLAST. The alignment results showed that the designed sgRNA spacer sequence had 100% coverage within the detected species and good inclusivity; the number of bases matched across species was <15 nt, and the specificity was good. The sgRNAs used are shown in Table 3.

[0053]

[0054] 2.2.2, sgRNA performance verification 2.2.2.1, PCR primer design According to the position of the designed sgRNA, the region within 100 bp upstream and downstream of the sgRNA was selected to design PCR primers (see Table 4). The primers were designed using commonly used software for primer design, and the primer length was controlled at 20-35 bp. If there are multiple sgRNA design sites within the same target fragment, the PCR amplification product (fragment length controlled within 500 bp) will contain all the sgRNA binding sites, so that all sgRNAs can be verified under the same template.

[0055]

[0056] 2.2.2.2, template amplification verification Plasmid / template DNA and corresponding primers were used for PCR amplification to obtain high-concentration PCR products. The specific amplification system is shown in Table 5, and the corresponding PCR reaction program is shown in Table 6.

[0057]

[0058]

[0059] The amplification products of the target to be tested and the blank control were detected by Qubit double-stranded DNA nucleic acid fluorescent dye concentration. The concentration of the target to be tested should be higher than 2 ng / μL, and there should be a clear distinction from the concentration of the blank control.

[0060] 2.2.2.3, sgRNA verification The CRISPR system was prepared according to the system in Table 7.

[0061]

[0062] Take 1 μL of about 10-100 ng PCR amplification product and mix with the above CRISPR system, set 43℃ reaction for 15 min in qPCR instrument, collect FAM fluorescence signal every minute. According to the curve change of fluorescence signal, the performance or specificity of sgRNA is preliminarily verified. The endpoint of fluorescence value curve of target amplification product and negative control should be significantly different.

[0063] The calculation formula of fluorescence intensity growth rate is as follows: .

[0064] Wherein, Fluorescence Slope represents the fluorescence growth rate, Rn represents the fluorescence signal of the nth minute, R1 represents the fluorescence signal of the first minute. Fluorescence Slope greater than 0.5 is positive, and Fluorescence Slope of negative group should not exceed 0.2.

[0065] 2.3, primer design and verification 2.3.1, RPA primer design In the detection of target range, select 30-35 base length fragments as RPA primer candidates; in order to ensure stability and specificity, the GC content of RPA primer should be between 40%-60%, and the RPA amplification fragment is usually 100-200 bp. In this project, the upstream and downstream primers were designed according to the primer design principle, and the NCBI primer-BLAST was used to verify the amplification coverage and specificity.

[0066] Based on the above design principles, RPA primers shown in Table 8 were designed for each plasmid / template for subsequent synthesis and verification experiments.

[0067]

[0068] 2.4, chip verification 2.4.1, chip detection process Nucleic acids and bacterial liquid are used for verification in this part.

[0069] Using the system determined in 3.2, the target nucleic acid sample is verified. First, the sample is quickly extracted, and then the sample lysate is diluted 8 times to prepare the RPA system. After mixing evenly, the chip is detected, and the fluorescence signal is judged according to the shooting.

[0070] The test process of the chip is shown in Table 9, and the total detection time is about 28 min.

[0071]

[0072] 3, experimental results 3.1, Screening results of sgRNA The fluorescence intensity of the amplification product of each sgRNA and the corresponding template was determined by CRISPR reaction. The amplification curve and fluorescence growth rate analysis results are shown in Figure 1 , and the results of fluorescence value change are shown in Table 10. The analysis results show that the BG target Bg-sgRNA has good effect.

[0073]

[0074] 3.2, Screening results of RPA primers Different combinations of RPA primers were screened using nucleic acid samples diluted to 10 ng / μL, and two-step CRISPR reverse reaction was used. The first step was RPA amplification reaction, and then the RPA amplification product was mixed with the CRISPR reaction solution for CRISPR reaction.

[0075] The change of Fluorescence Slope of the target amplification product within 15 min should be more than 0.5 to be judged as effective detection; the change of raw fluorescence value of negative control and blank control within 15 min should be less than 0.2.

[0076] The effective amplification rate of each primer at each dilution gradient is shown in Table 11.

[0077] The analysis results show that: All primers designed for BG target can achieve amplification of the sample. After comparing the performance, the RPA-BG-F3 / R4 primer pair with better amplification performance is selected for the next step of sensitivity verification. Figure 2

[0078]

[0079] 3.3, Construction and verification of detection system 3.3.1, Sensitivity verification of detection system The genomic template was diluted by gradient, and the genomic addition amount in the unit reaction was set to contain 156 pg, 15.6 pg, 7.8 pg, 1.56 pg, 0.78 pg, 0.156 pg, and 0.078 pg, which was adjusted according to the effect of different targets. The primers and sgRNA combination determined in 3.2 and the same CRISPR system were used to verify the sensitivity of the target. Each gradient was repeated for 3 times of experimental verification.

[0080] The amplification curve and fluorescence signal growth rate of sensitivity verification of each target are shown as follows. BG( Figure 3 ), and the lowest can detect 0.156 pg of nucleic acid sample.

[0081] ​According to the significance analysis of the sensitivity verification experiment results, the detection limit of the primer and the corresponding target can be preliminarily judged as shown in Table 12.

[0082]

[0083] According to the relationship between the genome copy number and the genome quality in the previous study, the BG sensitivity is about 5.7 copies / T.

[0084] 3.3.2, detection system specificity verification 5 cross-species were used for target specificity testing, with the target species as the positive control, the cross-species as the specificity verification test group, and water as the negative control template. The primers and sgRNA combinations determined in 3.2, and the same CRISPR system were used to verify the specificity of the target. Each cross-reaction was verified twice.

[0085] The results of the specificity verification of the BG target are shown in Figure 4 The verification results show that the BG target detects BG samples as positive, and the detection results of the 5 cross-species are negative, indicating that the BG target has good specificity in the cross-species.

[0086] 3.3.3, detection system sample compatibility test The nucleic acid was diluted to 10 ng / μL for this part of the verification.

[0087] Using the existing detection system, the actual sample of the target nucleic acid was verified. Each actual sample was verified twice.

[0088] The amplification curves and fluorescence signal growth rates of different subtypes of genomic samples are shown below. BG target can be compatible with BG2, BG3 subtypes (see Figure 5 ).

[0089] 3.4, development of bacterial liquid sample rapid extraction process 3.4.1, evaluation of the interference of the rapid extraction process on the system The interference of the target genome test rapid extraction process on the detection system was evaluated, with the original sample mixture (hereinafter referred to as the original solution) as the positive control, and the rapid extraction process sample as the test group. The primers and sgRNA combinations determined in 3.2, and the same CRISPR system were used for verification. Each cross-reaction was verified twice.

[0090] The verification results of the BG target ( Figure 6 ) show that the original solution detection is positive, and the detection effect of the rapid extraction sample is similar to that of the original solution, without obvious reduction.

[0091] 3.4.2, feasibility verification of bacteria liquid rapid extraction system The bacteria liquid was diluted 10 times and 100 times for this part of the verification.

[0092] Using the existing detection system, the target target bacteria liquid sample was verified. Each actual sample was verified twice.

[0093] The amplification curve and fluorescence signal growth rate of different concentrations of bacteria liquid samples are shown below. BG target bacteria liquid samples of two concentrations can be detected Figure 7 ; Overall, the rapid extraction process can be used to detect bacteria liquid.

[0094] 3.4.3, bacteria liquid performance verification of non-rapid extraction system Test with bacteria liquid sample 1E6 CFU / mL. Use the primer, template and sgRNA combination determined in 3.2, and the same CRISPR system. Take the rapid extraction system as a positive control, test the bacteria lysis effect of the non-rapid extraction process system (normal saline), and each test is verified twice.

[0095] The detection effect of normal saline lysis bacteria liquid is shown below, BG target ( Figure 8 ) cannot be detected with normal saline lysis, and the effect is obviously compared with the rapid extraction process system, which further illustrates that the rapid extraction process has obvious nucleic acid release effect on bacteria liquid.

[0096] 3.4.4, sensitivity verification of bacteria liquid rapid extraction process Gradient dilution of bacteria liquid, gradient setting includes 1E6 CFU / mL, 1E5 CFU / mL, 1E4 CFU / mL, 1E3 CFU / mL, 1E2 CFU / mL, 1E1 CFU / mL, 1 CFU / mL, corresponding to single reaction bacteria number respectively 156 CFU, 15.6 CFU, 1.56 CFU, 0.156 CFU, 0.0156 CFU, 0.00156 CFU, 0.000156 CFU, adjust according to different target effect. Use the primer, template and sgRNA combination determined in 3.2, and the same CRISPR system to verify the sensitivity of the target bacteria liquid. Each gradient is verified twice.

[0097] The amplification curve and fluorescence signal growth rate of bacteria liquid sensitivity verification are shown below, the minimum BG target can detect 15.6 CFU / T bacteria liquid sample ( Figure 9 ).

[0098] 3.5, chip detection results 3.5.1, initial chip detection results The nucleic acid sample was diluted to a concentration of 10 ng / μL. First, the rapid extraction process was tested on the chip, and the verification results are as follows Figure 10 BG target genome was not detected.

[0099] 3.5.2, Chip test BG target primer pair The nucleic acid sample was diluted to 10 ng / μL. The four pairs of RPA primers screened in 3.2 with good effect: RPA-BG-F1 / R3, RPA-BG-F1 / R4, RPA-BG-F2 / R4, RPA-BG-F3 / R4 were tested on the chip, and a two-step CRISPR reaction was used. The first step was RPA amplification, and then the RPA amplification product was mixed with the CRISPR reaction solution for CRISPR reaction. The fluorescence signal was used to determine the positive and negative results.

[0100] The verification results of different primer pairs of BG target are as follows Figure 11 RPA-BG-F1 / R3, RPA-BG-F2 / R4, RPA-BG-F3 / R4 were not detected, and RPA-BG-F1 / R4 could detect positive signals.

[0101] 3.5.3, Chip confirmation of BG target detection effect The BG target nucleic acid sample was diluted to 10 ng / μL, and the target primers and sgRNA screened in 3.2 were used for verification, and the BG primer pair was replaced with RPA-BG-F1 / R4 for chip testing. A two-step CRISPR reaction was used. The first step was RPA amplification, and then the RPA amplification product was mixed with the CRISPR reaction solution for CRISPR reaction. After completing the chip process, the results were interpreted according to the brightness change of the detection hole. In the chip setting, hole 1 corresponds to the BG target.

[0102] The verification results are as follows Figure 12 The BG target detection system corresponds to hole 1 on the chip, and it can be seen that only the sample is the BG sample when it is positive.

[0103] 3.5.4, Sensitivity of target bacteria liquid rapid extraction detection under chip process The bacteria liquid or genomic sample was gradient diluted, and the target primers screened in 3.2 were used for verification, and a two-step CRISPR reaction was used. The first step was RPA amplification, and then the RPA amplification product was mixed with the CRISPR reaction solution for CRISPR reaction. The algorithm program of the instrument was used to distinguish between positive and negative results (holes with boxes outside are positive holes).

[0104] The BG target bacteria liquid chip detection results are as follows Figure 13The BG target can be detected in the bacterial liquid after the rapid extraction process on the chip, the whole process is completed, and the minimum detectable bacterial liquid sample is 15.6 CFU.

[0105] In summary, the present application develops an isothermal amplification detection method based on RPA and CRISPR for BG target, which breaks away from the dependence on complex instrument equipment platform of traditional molecular biology method, greatly simplifies the detection platform; through system establishment and verification, the overall detection time of the target is shortened to within half an hour, and there is still room for further reduction in the future; at the same time, the sensitivity, specificity and POCT platform of the target detection technology are verified, and the results show that the developed detection method has good sensitivity and specificity, and the actual sample verification results are consistent with the results of the gold standard detection method. The detailed detection limit and verification data of the target are as follows: The BG target sgRNA is determined by Bg-sgRNA, which can be tested by primer RPA-BG-F3R4, the minimum detectable genome is 0.156 pg per reaction, which is equivalent to about 5.7 copies per reaction, and is specific in BG-CK1-BG-CK5. The minimum detectable bacterial liquid is 15.6 CFU / T by the rapid extraction process combined with the detection system, and the minimum detectable bacterial liquid on the chip is 15.6 CFU / T.

Claims

1. A reagent combination for detecting Burkholderia glumae, characterized by, comprising a specific RPA primer pair and an 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 is capable of targeting a specific sequence in the product amplified by the RPA primer pair, and its nucleotide sequence is as shown in SEQ ID NO.

3.

2. A kit for detecting Selenophoma thiveralensis, characterized by comprising the polynucleotide of claim 1. comprising: (1) the reagent combination of claim 1; (2) a Cas12b protein; (3) a ssDNA reporter molecule, which is labeled with a fluorescent reporter group at one end and a quenching group at the other end.

3. A method for detecting Magnaporthe graminea for non-diagnostic purposes, characterized by, comprising the following steps: S1, using the specific RPA primer pair in the reagent combination of claim 1 to perform RPA reaction with the nucleic acid of the sample to be tested as a template; S2, CRISPR detection reaction: mixing the amplification product with the sgRNA, Cas12b protein and single-stranded DNA fluorescent reporter molecule in the reagent combination of claim 1 and performing constant temperature incubation; S3, detecting the fluorescence signal, and determining whether the sample to be tested contains the rice sheath blight fungus according to the fluorescence signal.

4. The detection method according to claim 3, characterized in that, The RPA reaction is performed at 43℃ for 15 minutes; the constant temperature incubation is performed at 43℃ for 10 minutes.

5. The detection method according to claim 3, 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 comprises a RPA reaction chamber and a CRISPR detection chamber which are mutually isolated and in which the RPA primer pair and the sgRNA in the reagent combination of claim 1 are pre-disposed.

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