Rapid detection method for four pathogenic bacteria of corn based on RPA-CRISPR / Cas12b technology

Abstract

The invention discloses a rapid detection method for four pathogenic bacteria of corn based on an RPA-CRISPR / Cas12b technology, and belongs to the technical field of nucleic acid detection. The method provides a specific RPA (recombinase polymerase amplification) primer pair and an sgRNA (single guide ribonucleic acid) sequence aiming at four quarantine pests such as CMN (fusarium wilt disease), Pss (bacterial fusarium wilt disease), MCMV (maize chlorotic mottle virus) and MDMV (maize dwarf mosaic virus). Recombinase polymerase amplification (RPA) and CRISPR / Cas12b detection technologies are combined and are integrated on a centrifugal micro-fluidic chip, so that high-sensitivity and high-specificity detection on multi-target nucleic acid is realized, integrated detection of'sample input-result output 'can be completed within 30 minutes, uncovering operation is effectively avoided, the risk of aerosol pollution is greatly reduced, and the method is suitable for industrial production. The method is suitable for port quarantine and field on-site rapid detection, and an efficient technical means is provided for early diagnosis and prevention and control of corn diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nucleic acid detection, and particularly relates to a rapid detection method for corn interior wilt fungus (CMN), corn bacterial wilt fungus (Pss), corn chlorotic mottle virus (MCMV) and corn dwarf mosaic virus (MDMV) based on RPA-CRISPR / Cas12b technology. BACKGROUND

[0002] Although corn is an important food and feed crop in China, corn interior wilt fungus (CMN), corn bacterial wilt fungus (Pss), corn chlorotic mottle virus (MCMV) and corn dwarf mosaic virus (MDMV) and other varieties of quarantine pathogens seriously harm the growth of corn and cause yield loss or even absolute loss. Therefore, developing a rapid, accurate and practical detection technology is of great significance for corn disease control and port quarantine.

[0003] At present, the detection of pathogenic bacteria 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 good specificity, but it is seriously dependent on a thermal cycler, is complicated to operate and time-consuming, and is difficult to apply to on-site rapid detection. The isothermal amplification technologies such as LAMP and RPA overcome the dependence on temperature cycling to some extent, significantly shorten the amplification time and provide a possibility for the development of on-site detection.

[0004] However, these methods still have obvious limitations: first, most of the detection still needs to be opened for electrophoresis or test strip detection after completion, which increases the risk of aerosol pollution and easily leads to false positive results, and the reliability of multiplex detection is insufficient; second, the whole process involves multiple steps of operation, which is difficult to integrate, limiting its application effect in on-site environments such as ports and fields.

[0005] Therefore, it is urgent to establish a new multi-target detection method with high sensitivity, high specificity, simple operation and suitable for on-site rapid detection to overcome the bottleneck of existing technologies in practical application. SUMMARY

[0006] The application provides a corn pathogen rapid detection method based on RPA-CRISPR / Cas12b technology, which can be used for high-sensitivity and high-specificity detection of four quarantine harmful organisms, i.e., Clavibacter michiganensis subsp. nebraskense (CMN), Pantoea stewartii subsp. Stewartia (Pss), Maize chlorotic mottle virus (MCMV) and Maize dwarf mosaic virus (MDMV). The method combines RPA and CRISPR detection technology, and is integrated into a microfluidic chip and a portable detection device, effectively solving the problems of existing technology, such as dependence on large equipment, complex operation, long time consumption and difficulty in realizing on-site multi-target detection, and is suitable for rapid screening and diagnosis in the field and at the port.

[0007] To achieve the above-mentioned application purposes, the application adopts the following technical solutions: The application provides a reagent combination for simultaneously detecting multiple corn pathogenic bacteria, wherein the pathogenic bacteria are Clavibacter michiganensis subsp. nebraskense (CMN), Pantoea stewartii subsp. Stewartia (Pss), Maize chlorotic mottle virus (MCMV) and Maize dwarf mosaic virus (MDMV); and the reagent combination comprises specific RPA primer pairs and sgRNA. When the pathogenic bacteria are CMN, the RPA primer pairs are composed 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; and the sgRNA can target a specific sequence in the product amplified by the RPA primer pairs, and has a nucleotide sequence as shown in SEQ ID NO. 3. When the pathogenic bacteria are Pss, the RPA primer pairs are composed of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 4 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 5; and the sgRNA can target a specific sequence in the product amplified by the RPA primer pairs, and has a nucleotide sequence as shown in SEQ ID NO. 6. When the pathogenic bacteria are MCMV, the RPA primer pairs are composed of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 8; and the sgRNA can target a specific sequence in the product amplified by the RPA primer pairs, and has a nucleotide sequence as shown in SEQ ID NO. 9. When the pathogenic bacteria is MDMV, the RPA primer pair consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 10 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 11; the sgRNA can target a specific sequence in the product amplified by the RPA primer pair, and has a nucleotide sequence as shown in SEQ ID NO. 12.

[0008] The application also provides a kit for simultaneously detecting multiple pathogenic bacteria of corn, comprising: (1) the reagent combination; (2) Cas12b protein; (3) ssDNA reporter molecule, which is labeled with a fluorescent reporter group at one end and a quenching group at the other end.

[0009] The application also provides a method for simultaneously detecting multiple pathogenic bacteria of corn for non-diagnostic purposes, comprising the following steps: S1, using the nucleic acid of the sample to be tested as a template, performing RPA reaction using the corresponding specific RPA primer pair in the reagent combination; S2, CRISPR detection reaction: mixing the amplification product with the corresponding sgRNA, Cas12b protein and single-stranded DNA fluorescent reporter molecule in the reagent combination and performing constant temperature incubation; S3, detecting the fluorescence signal, and determining whether the sample to be tested contains Phialophora gregata, Fusarium verticillioides, maize chlorotic mottle virus or maize dwarf mosaic virus according to the fluorescence signal.

[0010] Further, the RPA reaction is performed at 43℃ for 15 minutes; and the constant temperature incubation is performed at 43℃ for 10 minutes.

[0011] Further, the RPA reaction, CRISPR detection reaction and fluorescence 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 corresponding RPA primer pair and sgRNA in the reagent combination are pre-disposed. Advantages

[0012] (1) The RPA isothermal amplification technology adopted in the application does not require complex temperature cycling, and in combination with the high-efficiency cutting reaction of CRISPR / Cas12b, nucleic acid amplification can be completed within 15 minutes, and detection can be completed within 10 minutes, and the whole process from sample addition to obtaining results takes no more than 30 minutes, which is much faster than traditional PCR technology (usually 1.5-2 hours), greatly improves the detection efficiency, and meets the needs of emergency monitoring and on-site rapid decision-making.

[0013] (2) The specific RPA primer and sgRNA combination designed by the application, and the double recognition and signal amplification of the target sequence by the CRISPR-Cas12b system, make the detection method have high sensitivity and specificity for the detection of corn northern wilt fungus (CMN), corn bacterial wilt fungus (Pss), corn chlorotic mottle virus (MCMV) and corn dwarf mosaic virus (MDMV), which is much better than the conventional PCR method. And no false positive signal appears in the cross reaction test, which ensures the accuracy and reliability of the detection result.

[0014] (3) The application not only provides a detection scheme for a single pathogenic bacterium, but more importantly, through the multi-chamber design of the microfluidic chip, CMN, Pss, MCMV and MDMV can be simultaneously and parallelly detected on one chip, and the screening of multiple potential threat factors can be completed at one time, which greatly improves the throughput and efficiency of detection.

[0015] (4) The application simplifies the complex multi-step operation into one-time sample addition through reagent pre-freeze drying and chip design. The detection process is automatically completed by a portable device, and does not require a complex thermal cycler and professional operators, which greatly reduces the use threshold and is suitable for limited resources such as grassroots units, ports and field sites. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The application employs a centrifugal microfluidic detection platform, and the three-dimensional structure schematic diagram thereof is shown.

[0017] Figure 2 The microfluidic chip structure decomposition and chamber layout schematic diagram is shown, mainly showing the sample bin, RPA reaction chamber, CRISPR detection chamber and connecting flow channel.

[0018] Figure 3 The microfluidic chip in the working process, the reaction liquid flows through different chambers in the preset time sequence under the driving of centrifugal force, and the flow process schematic diagram is shown.

[0019] Figure 4 The performance screening result graph of three candidate sgRNAs of MDMV target in the embodiment of the application is shown; (A) is the fluorescence change rate; (B) is the fluorescence curve.

[0020] Figure 5 The performance screening result graph of three candidate sgRNAs of MCMV target in the embodiment of the application is shown; (A) is the fluorescence change rate; (B) is the fluorescence curve.

[0021] Figure 6 The performance screening result graph of three candidate sgRNAs of Pss target in the embodiment of the application is shown; (A) is the fluorescence change rate; (B) is the fluorescence curve.

[0022] Figure 7 Figure for performance screening result of three candidate sgRNAs of CMN target in the embodiment of the present application; (A) is fluorescence change rate; (B) is fluorescence curve.

[0023] Figure 8 Figure for performance screening result of different RPA primer combinations of MDMV target in the embodiment of the present application; (A) is fluorescence change rate; (B) is fluorescence curve of each primer combination under high concentration template (1E4 copy number / reaction); (C) is fluorescence curve of each primer combination under low concentration template (1E3 copy number / reaction).

[0024] Figure 9 Figure for performance screening result of different RPA primer combinations of CMN target in the embodiment of the present application; (A) is fluorescence change rate; (B) is fluorescence curve of each primer combination under high concentration template (1E4 copy number / reaction); (C) is fluorescence curve of each primer combination under low concentration template (1E3 copy number / reaction).

[0025] Figure 10 Figure for performance screening result of different RPA primer combinations of MCMV target in the embodiment of the present application; (A) is fluorescence change rate; (B) is fluorescence curve of each primer combination under high concentration template (1E4 copy number / reaction); (C) is fluorescence curve of each primer combination under low concentration template (1E3 copy number / reaction).

[0026] Figure 11 Figure for performance screening result of different RPA primer combinations of Pss target in the embodiment of the present application; (A) is fluorescence change rate; (B) is fluorescence curve of each primer combination under high concentration template (1E4 copy number / reaction); (C) is fluorescence curve of each primer combination under low concentration template (1E3 copy number / reaction).

[0027] Figure 12 Figure for sensitivity verification result of MDMV target in the embodiment of the present application; (A) is fluorescence change rate; (B) is fluorescence curve.

[0028] Figure 13 Figure for sensitivity verification result of Pss target in the embodiment of the present application; (A) is fluorescence change rate; (B) is fluorescence curve.

[0029] Figure 14 Figure for sensitivity verification result of CMN target in the embodiment of the present application; (A) is fluorescence change rate; (B) is fluorescence curve.

[0030] Figure 15 Figure for sensitivity verification result of MCMV target in the embodiment of the present application; (A) is fluorescence change rate; (B) is fluorescence curve.

[0031] Figure 16 Figure for sensitivity confirmation of Pss target near the detection limit in the embodiment of the application; (A) is the fluorescence change rate of 10 repeated detections; (B) is the fluorescence curve of high concentration template (5E2 copies / reaction); (C) is the fluorescence curve of low concentration template (1E2 copies / reaction).

[0032] Figure 17 Figure for sensitivity confirmation of MDMV target near the detection limit in the embodiment of the application; (A) is the fluorescence change rate of 10 repeated detections; (B) is the fluorescence curve of high concentration template (5E2 copies / reaction); (C) is the fluorescence curve of low concentration template (1E2 copies / reaction).

[0033] Figure 18 Figure for sensitivity confirmation of MCMV target near the detection limit in the embodiment of the application; (A) is the fluorescence change rate of 10 repeated detections; (B) is the fluorescence curve of high concentration template (5E2 copies / reaction); (C) is the fluorescence curve of low concentration template (1E2 copies / reaction).

[0034] Figure 19 Figure for sensitivity confirmation of CMN target near the detection limit in the embodiment of the application; (A) is the fluorescence change rate of 10 repeated detections; (B) is the fluorescence curve of high concentration template (5E2 copies / reaction); (C) is the fluorescence curve of low concentration template (1E2 copies / reaction).

[0035] Figure 20 Figure for detection effect of CRISPR detection system on four single target nucleic acids with 1E4 copies / reaction concentration on the microfluidic chip after drying treatment.

[0036] Figure 21 Figure for full-process closed tube detection effect of RPA primer and CRISPR detection system on four single target nucleic acids with 1E4 copies / reaction concentration on the microfluidic chip after drying treatment.

[0037] Figure 22 Figure for full-process closed tube detection effect of RPA primer and CRISPR detection system on four single target nucleic acids with 1E4 copies / reaction concentration on the microfluidic chip after drying treatment.

[0038] Figure 23 Figure for sensitivity confirmation of full-process closed tube detection of RPA primer and CRISPR detection system on four single target nucleic acids with 1E4 copies / reaction concentration on the microfluidic chip after drying treatment. DETAILED DESCRIPTION

[0039] In order to better understand the technical solutions in the present application, the present application will be further described in detail below in conjunction with embodiments.

[0040] The nucleotide sequences involved in the present application are as follows: SEQ ID NO. 1: The sequence shown is the nucleotide sequence of RPA primer MDMV-RPA-F3.

[0041] SEQ ID NO. 2: The sequence shown is the nucleotide sequence of RPA primer MDMV-RPA-R1.

[0042] SEQ ID NO. 3: The sequence shown is the nucleotide sequence of sgRNA target MDMV-sgRNA-3.

[0043] SEQ ID NO. 4: The sequence shown is the nucleotide sequence of RPA primer MCMV-RPA-F2.

[0044] SEQ ID NO. 5: The sequence shown is the nucleotide sequence of RPA primer MCMV-RPA-R1.

[0045] SEQ ID NO. 6: The sequence shown is the nucleotide sequence of sgRNA target MCMV-sgRNA-2.

[0046] SEQ ID NO. 7: The sequence shown is the nucleotide sequence of RPA primer Pss-RPA-F1.

[0047] SEQ ID NO. 8: The sequence shown is the nucleotide sequence of RPA primer Pss-RPA-R1.

[0048] SEQ ID NO. 9: The sequence shown is the nucleotide sequence of sgRNA target Pss-sgRNA-3.

[0049] SEQ ID NO. 10: The sequence shown is the nucleotide sequence of RPA primer CMN-RPA-F1.

[0050] SEQ ID NO. 11: The sequence shown is the nucleotide sequence of RPA primer CMN-RPA-R2.

[0051] SEQ ID NO. 12: The sequence shown is the nucleotide sequence of sgRNA target CMN-sgRNA-1.

[0052] SEQ ID NO. 13: The sequence shown is the nucleotide sequence of sgRNA target MDMV-sgRNA-1.

[0053] SEQ ID NO. 14: The sequence shown is the nucleotide sequence of sgRNA target MDMV-sgRNA-2.

[0054] SEQ ID NO. 15: The sequence shown is the nucleotide sequence of sgRNA target MCMV-sgRNA-1.

[0055] SEQ ID NO. 16: The sequence shown is the nucleotide sequence of sgRNA target MCMV-sgRNA-3.

[0056] SEQ ID NO. 17: The sequence shown is the nucleotide sequence of sgRNA target CMN-sgRNA-2.

[0057] SEQ ID NO. 18: The sequence shown is the nucleotide sequence of sgRNA target CMN-sgRNA-3.

[0058] SEQ ID NO. 19: The sequence shown is the nucleotide sequence of sgRNA target Pss-sgRNA-1 SEQ ID NO. 20: The sequence shown is the nucleotide sequence of sgRNA target Pss-sgRNA-2 SEQ ID NO. 21 : The sequence shown is the nucleotide sequence of PCR primer MDMV-PCR-F.

[0059] SEQ ID NO. 22: The sequence shown is the nucleotide sequence of PCR primer MDMV-PCR-R.

[0060] SEQ ID NO. 23: The sequence shown is the nucleotide sequence of PCR primer MCMV-PCR-F.

[0061] SEQ ID NO. 24: The sequence shown is the nucleotide sequence of PCR primer MCMV-PCR-R.

[0062] SEQ ID NO. 25: The sequence shown is the nucleotide sequence of PCR primer Pss-PCR-F.

[0063] SEQ ID NO. 26: The sequence shown is the nucleotide sequence of PCR primer Pss-PCR-R.

[0064] SEQ ID NO. 27: The sequence shown is the nucleotide sequence of PCR primer CMN-PCR-F.

[0065] SEQ ID NO. 28: The sequence shown is the nucleotide sequence of PCR primer CMN-PCR-R.

[0066] SEQ ID NO. 29: The sequence shown is the nucleotide sequence of RPA primer MDMV-RPA-F1.

[0067] SEQ ID NO. 30: The sequence shown is the nucleotide sequence of RPA primer MDMV-RPA-F2.

[0068] SEQ ID NO. 31 : The sequence shown is the nucleotide sequence of RPA primer MDMV-RPA-R2.

[0069] SEQ ID NO. 32: The sequence shown is the nucleotide sequence of RPA primer MDMV-RPA-R3.

[0070] SEQ ID NO. 33: The sequence shown is the nucleotide sequence of RPA primer CMN-RPA-F2.

[0071] SEQ ID NO. 34: The sequence shown is the nucleotide sequence of RPA primer CMN-RPA-F3.

[0072] SEQ ID NO. 35: The sequence shown is the nucleotide sequence of RPA primer CMN-RPA-R1.

[0073] SEQ ID NO. 36: The sequence shown is the nucleotide sequence of RPA primer CMN-RPA-R3.

[0074] SEQ ID NO. 37: The sequence shown is the nucleotide sequence of RPA primer MCMV-RPA-F1.

[0075] SEQ ID NO. 38: The sequence shown is the nucleotide sequence of RPA primer MCMV-RPA-F3.

[0076] SEQ ID NO. 39: The sequence shown is the nucleotide sequence of RPA primer MCMV-RPA-R2.

[0077] SEQ ID NO. 40: The sequence shown is the nucleotide sequence of RPA primer MCMV-RPA-R3.

[0078] SEQ ID NO. 41 : The sequence shown is the nucleotide sequence of RPA primer Pss-RPA-F2.

[0079] SEQ ID NO. 42: The sequence shown is the nucleotide sequence of RPA primer Pss-RPA-F3.

[0080] SEQ ID NO. 43: The sequence shown is the nucleotide sequence of RPA primer Pss-RPA-R2.

[0081] SEQ ID NO. 44: The sequence shown is the nucleotide sequence of RPA primer Pss-RPA-R3. Examples

[0082] 1. Experimental materials 1.1. Instruments and equipment SynSor portable multi-target rapid detection device, XS-D-001

Xunshi Biology

Shanghai Hongshi Medical Technology Co., Ltd.

Hangzhou Bozhi Technology Co., Ltd.

Thermo Fisher Scientific

Thermo Fisher Scientific

Hangzhou Ruicheng Instrument Co., Ltd.

[0083] 1.2. Reagents SynSor AaCas 12b (C2c1) (XS-R-002)

Xunshi Biology

Xunshi Biology

Xunshi Biology

Xunshi Biology

Xunshi Biology

Beijing Qikexing Biological Technology Co., Ltd.

Engelbrecht (Shanghai) Trade Co., Ltd.

[0084] 2. Experimental methods 2.1. sgRNA design and verification 2.1.1. sgRNA design According to the position in Corn Bacterial Blight (Pantoea stewartii subsp. stewartii)

Genbank index number: AJ311838.1 (1599-1895)

Genbank index number: AJ311838.1 (1599-1895)

Genbank index number: MK569413.1 (95-273)

Genbank index number: MZ326131.1 (342-821)

[0085]

[0086] 2.1.2, sgRNA performance verification 2.1.2.1, PCR primer design According to the position of the designed sgRNA, the region within 100bp upstream and downstream of the sgRNA was selected to design PCR primers, and the specific sequence information of the primers is shown in Table 2.

[0087] 2.1.2.2, template amplification verification According to the following system configuration solution, PCR amplification was carried out.

[0088] PCR amplification system: 1.1×Green MIX 42 μL; 10 μM upstream and downstream primers 2 μL each, template 2 μL, water 2 μL.

[0089] PCR reaction program: 98℃ pre-denaturation 2min; 98℃ denaturation 10s, 50℃ annealing 15s, 72℃ extension 10s, 30 cycles; finally 72℃ terminal extension 5min; 10℃ preservation 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.

[0090] 2.1.2.3 sgRNA Validation Prepare the CRISPR system according to Table 3.

[0091]

[0092] Mix 1 μL (approximately 10–100 ng) of PCR amplification product with the CRISPR system in Table 3, 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 sgRNA performance or specificity. The endpoint where the fluorescence curves of the target amplification product and the negative control stop rising should be significantly different.

[0093] The formula for calculating the fluorescence growth rate is as follows: .

[0094] Where Fluorescence Slop represents the fluorescence growth rate, Rn represents the fluorescence signal at minute n, and R1 represents the fluorescence signal of the negative control at minute 1.

[0095] 2.2 RPA Primer Design The design principles for RPA primers are as follows: within the target detection range, select fragments of 30-35 bases in length as RPA primer candidates; 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 these primer design principles, three upstream and three downstream primers were designed for RPA primer validation, and NCBI primer-BLAST was used to validate amplification coverage and specificity.

[0096] Based on the above principles, RPA-specific primers for four maize pathogens were designed, as shown in Table 4:

[0097] 2.3 Validation of the Cube Joint Testing System 2.3.1 Cube Platform and Chip Technology Principles SynsorCube-4X (hereinafter referred to as Cube) is a detection platform independently developed by Beijing Xunshi Technology Co., Ltd., capable of simultaneously detecting up to four targets and four samples. It utilizes technologies such as RPA amplification, CRISPR detection, a centrifugal microfluidic fully enclosed chip platform, and an intelligent image-based fluorescence detection system. Figure 1), and combined with reagent dry powder storage technology, attempts to realize a fully closed integrated nucleic acid rapid detection system, which can realize rapid identification of target and completely realize the detection mode of "sample in, result out".

[0098] SynsorCube-4X microfluidic chip (hereinafter referred to as Cube chip) is designed to distinguish sample loading channel, RPA amplification cavity and CRISPR detection cavity in physics (Figure 2), avoiding cross interference between the three, so that the performance of each step can be fully played out, greatly improving the accuracy and sensitivity of the detection system for nucleic acid target identification; In order to further reduce the complexity of the detection system, solve the problem that the centrifugal platform on the market is a differential centrifugal platform, the flow channel siphon valve design is introduced into the Cube chip, which can realize the precise transfer of liquid under different centrifugal processes (Figure 3) under the condition of constant speed of centrifugal module, further reducing the complexity and design cost of the whole detection system; As an important part of the detection system, the supporting equipment is an intelligent photographing fluorescence detection system that can perform multi-step centrifugation, bath reaction, fluorescence signal collection, analysis and output, realizing accurate reception and interpretation of the final detection fluorescence signal.

[0099] 2.3.2, Cube platform reaction system configuration 2.3.2.1, Cube platform CRISPR system configuration and preparation process Due to the particularity of Cube platform, in-situ verification of CRISPR system is needed before verification and development. The CRISPR in-situ system is shown in Table 5.

[0100]

[0101] After the above CRISPR system is prepared, 10 μL of the system is added to reaction chamber 2, and the chip is placed in a 50℃ oven, and the system is dried for 1.5 h; After drying is completed, the chip is taken out, and after being covered with a film, it is placed in a sealed aluminum foil bag and stored in a low humidity environment.

[0102] 2.3.2.2, RPA system configuration in CRISPR system verification stage of Cube platform This part tests the preparation of RPA amplification system of target. The final concentration of each component in the RPA system in this process is the same as that in section 3.2, and the addition amount of RPA freeze-dried ball in the system is 1. The addition amount of each component in the RPA system in this process is shown in Table 6.

[0103]

[0104] After mixing all the reaction systems in RPA, transfer all the systems to the loading hole of the Cube chip, then seal the film tightly, and place the Cube chip in the Cube instrument. Start the instrument on the mobile phone for detection. After the reaction process is completed, the Cube chip detection results can be viewed on the mobile phone, and the detection results can be read.

[0105] 2.3.2.3, Cube platform whole process system configuration and preparation process The CRISPR reaction system configuration and preparation process in the whole process chip are shown in 2.3.2.1.

[0106] The amount of each component in the RPA system in this process is shown in Table 7, and the components of the loading solution in the Cube platform whole process are shown in Table 8.

[0107]

[0108]

[0109] In the preparation of the whole process chip, the CRISPR system in Table 5 and the RPA primer system in Table 7 are placed in reaction chamber 2 and reaction chamber 1 respectively, and the chip is placed in a 50℃ oven, and the system is dried for 1.5h; after drying is completed, the chip is taken out, and after being covered with a film, it is placed in a sealed aluminum foil bag and stored in a low humidity environment.

[0110] After mixing all the loading solution systems, transfer them to the loading hole of the Cube chip, then seal the film tightly, and place the Cube chip in the Cube instrument. Start the instrument on the mobile phone for detection. After the reaction process is completed, the Cube chip detection results can be viewed on the mobile phone, and the detection results can be read.

[0111] 2.3.3, chip detection process The prepared nucleic acid sample is used for verification in this part.

[0112] The test process of the chip is shown in Table 9, and the total detection time is about 28min.

[0113]

[0114] 3, experimental results 3.1, sgRNA screening results The fluorescence intensity of the amplification product of each sgRNA and the corresponding template is determined by CRISPR reaction. The fluorescence curve and fluorescence growth rate trend are shown in Figures 4-7, the results of fluorescence growth rate are shown in Table 10. The analysis results show that: MDMV target MDMV-sgRNA-3 (SEQ ID NO. 3), MCMV target MCMV-sgRNA-2 (SEQ ID NO. 6), Pss target Pss-sgRNA-3 (SEQ ID NO. 9), CMN target CMN-sgRNA-1 (SEQ ID NO. 12) have better effect, which can be used for subsequent RPA primer screening.

[0115]

[0116] 3.2, RPA primer screening results Plasmid samples were diluted to 1E4 copies per reaction and 1E3 copies per reaction for RPA primer screening of different combinations, using two-step RPA-CRISPR reaction. In this process, the fluorescence curve and fluorescence growth rate trend of the detection effect of different primer groups Figures 8-11 , the effective amplification rate of each primer at each dilution gradient is shown in Tables 11, 12, 13, and 14.

[0117]

[0118]

[0119]

[0120]

[0121] The MDMV target primer verification results show that MDMV-RPA-F1 / R1 and MDMV-RPA-F3 / R1 have strong fluorescence signals under high concentration (1E4 copies per reaction) and low concentration sample (1E3 copies per reaction) conditions Figure 8 ), but MDMV-RPA-F1 / R1 (SEQ ID NO. 29 / SEQ ID NO. 2) NTC also has certain false positive fluorescence signal, so MDMV-RPA-F3 / R1 (SEQ ID NO. 1 / SEQ ID NO. 2) is selected for subsequent performance verification.

[0122] The CMN target primer verification results show that CMN-RPA-F1 / R2 has strong fluorescence signals under high concentration (1E4 copies per reaction) and low concentration sample (1E3 copies per reaction) conditions (Figure 9), so CMN-RPA-F1 / R2 (SEQ ID NO. 10 / SEQ ID NO. 11) is selected for subsequent performance verification experiments.

[0123] MCMV target primer verification results show that MCMV-RPA-F2 / R1 has strong fluorescence signal under high concentration (1E4 copy number / reaction) and low concentration sample (1E3 copy number / reaction) conditions (Figure 10), so MCMV-RPA-F2 / R1 (SEQ ID NO. 4 / SEQ ID NO. 5) is selected for subsequent performance verification experiments.

[0124] Pss target primer verification results show that Pss-RPA-F1 / R1 and Pss-RPA-F2 / R1 have strong fluorescence signals under high concentration (1E4 copy number / reaction) and low concentration sample (1E3 copy number / reaction) conditions (Figure 11), but Pss-RPA-F2 / R1 (SEQ ID NO. 41 / SEQ ID NO. 8) NTC also has a certain false positive fluorescence signal, so Pss-RPA-F1 / R1 (SEQ ID NO. 7 / SEQ ID NO. 8) is finally selected for subsequent performance verification experiments.

[0125] 3.3, Test system construction and verification 3.3.1, Sensitivity verification of detection system The plasmid template is diluted down to contain 1E4 copy number / reaction, 1E3 copy number / reaction, 1E2 copy number / reaction, 1E1 copy number / reaction, and 1E0 copy number / reaction. The primers and sgRNA combinations determined in 3.2 are used, and the same CRISPR system is used to verify the sensitivity of the target. Each gradient is verified by 2 experimental repeats.

[0126] In this part, the amplification curves and fluorescence growth rates of the sensitivity verification of each target are shown as follows: Figure 12 1E1 copy number / reaction can be detected, Pss ( Figure 13 ), CMN ( Figure 14 ), and MCMV ( Figure 15 ) 1E2 copy number / reaction can be detected.

[0127] 3.3.2, Sensitivity confirmation of detection system After completing the sensitivity verification, 10 experimental repeats are continued near the target detection limit to confirm the sensitivity of the target. According to the sensitivity of each target point verified in the early stage, Pss and MDMV targets in this part use 5E1, 1E2 copy number / reaction concentration group to confirm the sensitivity, MCMV and CMN targets use 5E2, 1E2 copy number / reaction. The amplification curves and fluorescence signal growth rates of the detection limit verification of each target are shown in Figures 16-19 .

[0128] The sensitivity verification and sensitivity confirmation results of the four-target RPA-CRISPR detection system showed that the four targets exhibited good stability in the detection limit and the 10 repeats near the detection limit. Therefore, the detection limit of Pss and MDMV targets was 5E1 copy number / reaction, the detection limit of CMN was 1E2 copy number / reaction, and the detection limit of MCMV target was 5E2 copy number / reaction. The detection sensitivity was better than that of the common method at the present stage.

[0129] 3.3.3, single-target chip CRISPR reagent in-situ drying feasibility verification The previously synthesized plasmid samples were diluted, and the loading concentration was 1E4 copy number / reaction. The chip preparation process in this process is shown in 2.3.2.1, and the loading system is shown in 2.3.2.2. Each chip corresponds to one target. The verification results of each target are shown in Figure 20 The test results of this round showed that the four targets could produce strong fluorescence signals after CRISPR in-situ, indicating that the CRISPR in-situ performance of the four targets met the subsequent test requirements.

[0130] 3.3.4, single-target chip RPA and CRISPR double system in-situ drying feasibility verification Based on the previous verification of CRISPR detection single system in-situ, RPA and CRISPR double system in-situ drying test was further carried out. In this part, nucleic acid samples diluted to 1E4 copy number / reaction were used, and the chip test double system drying process is shown in 2.3.2.3. The CRISPR, RPA reagent in-situ system and the cube chip loading liquid system are shown in Table 5, Table 6 and Table 8. The test results of this round are shown in Figure 21 The test results of this round showed that the primer combinations developed in the previous stage had good detection results in the RPA and CRISPR double system in-situ drying test. The four targets double system in-situ could achieve positive detection, indicating that the subsequent multi-target cube chip verification could be carried out 3.3.5, MDMV, Pss, CMN, MCMV multi-target chip feasibility verification Based on the previous verification, nucleic acid samples were diluted to 1E4 copy number / reaction for chip testing. The selected primer combinations MDMV-RPA-F3 / R1, Pss-RPA-F1 / R1, CMN-RPA-F1 / R2 and MCMV-RPA-F2 / R1 were used to confirm the detection effect with the cube platform full process system configuration and preparation process of 2.3.2.3. The detection performance confirmation results are shown in Figure 22The experimental verification results show that the primer combination of the four targets matched with the CRISPR process can output strong positive signals in the Cube chip platform. At the same time, the four-target chip has only one sample added, and only the corresponding hole can produce a positive signal, indicating that the designed RPA-CRISPR detection process has good specificity in the four targets.

[0131] 3.3.6, MDMV, Pss, CMN, MCMV multi-target chip sensitivity verification On the basis of the feasibility verification of the multi-target chip, the sensitivity verification of the multi-target chip was continued, and 1E4, 1E3, 1E2, 1E1 copy number / reaction samples were selected for Cube platform sensitivity verification. The verification results are shown in Figure 23 The verification results show that the detection sensitivity of all target chips can reach 1E2 copy number / reaction.

[0132] In summary, the present application develops an isothermal amplification detection method based on RPA and CRISPR for MDMV, Pss, CMN, MCMV disease targets commonly found in corn, which breaks away from the dependence on complex instrument equipment platform of traditional molecular biology methods, greatly simplifying the detection platform; through system establishment and verification, the overall detection time of the target is shortened to less than 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 theoretical results. The target detection limit and verification data are as follows: (1) The MDMV target sgRNA is determined by MDMV-sgRNA-3, which can be combined with primers MDMV-RPA-F3 / R1 for testing, and the minimum detection limit is 5E1 copy number / reaction, and specific in Pss, CMN, MCMV; The RPA primer set of the in-tube system of this target can be used for Cube chip testing, and the sensitivity under the Cube chip detection process can reach 1E2 copy number / reaction; (2) The Pss target sgRNA is determined by Pss-sgRNA-3, and the combination of primers Pss-RPA-F1 / R1 has good effect, and the minimum detection limit is 5E1 copy number / reaction, and specific in MDMV, CMN, MCMV cross species; The RPA primer set of the in-tube system of this target can be used for Cube chip testing, and the sensitivity under the Cube chip detection process can reach 1E2 copy number / reaction; (3) The CMN target sgRNA was determined as CMN-sgRNA-1, and the detection effect was better when combined with CMN-RPA-F1 / R2, with a minimum detection limit of 1E2 copies / reaction, and the target in-vitro system RPA primer set could be used for Cube chip testing, and the sensitivity under the Cube chip detection process could reach 1E2 copies / reaction; (4) The MCMV target sgRNA was determined as MCMV-sgRNA-2, and the detection effect was better when combined with MCMV-RPA-F2 / R1, with a minimum detection limit of 5E2 copies / reaction, and the target in-vitro system RPA primer set could be used for Cube chip testing, and the sensitivity under the Cube chip detection process could reach 1E2 copies / reaction.

Claims

1. A reagent combination for simultaneous detection of multiple pathogens of corn, characterized in that, The pathogenic bacteria are Clavibacter michiganensis subsp. nebraskense (CMN), Pantoea stewartii subsp. Stewartia (Pss), Maize chlorotic mottle virus (MCMV) and Maize dwarf mosaic virus (MDMV); the reagent combination comprises specific RPA primer pairs and sgRNA; When the pathogenic bacteria are CMN, 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 has a nucleotide sequence as shown in SEQ ID NO. 3; When the pathogenic bacteria are Pss, the RPA primer pair consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 4 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 5; the sgRNA can target a specific sequence in the product amplified by the RPA primer pair, and has a nucleotide sequence as shown in SEQ ID NO. 6; When the pathogenic bacteria are MCMV, the RPA primer pair consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 8; the sgRNA can target a specific sequence in the product amplified by the RPA primer pair, and has a nucleotide sequence as shown in SEQ ID NO. 9; When the pathogenic bacteria are MDMV, the RPA primer pair consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 10 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 11; the sgRNA can target a specific sequence in the product amplified by the RPA primer pair, and has a nucleotide sequence as shown in SEQ ID NO.

12.

2. A kit for simultaneous detection of multiple pathogens of corn, characterized in that, Comprise: (1) the reagent combination of claim 1; (2) Cas12b protein; (3) ssDNA reporter molecule, one end of which is labeled with a fluorescent reporter group, and the other end is labeled with a quenching group.

3. A method for detecting a plurality of pathogens of corn for non-diagnostic purposes, characterized by, Comprise the following steps: S1, using the nucleic acid of the sample to be tested as a template, performing RPA reaction using the corresponding specific RPA primer pair in the reagent combination of claim 1; S2, CRISPR detection reaction: mixing the amplification product with the corresponding 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 judging whether the sample contains the C. nigrum, the B. emersonii, the M. acuta, the M. acuta or the M. acuta according to the fluorescence signal.

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

5. The detection method according to claim 3, characterized in that, The RPA reaction, the CRISPR detection reaction and the fluorescence signal detection are all automatically completed on a centrifugal microfluidic chip; the centrifugal microfluidic chip comprises mutually isolated RPA reaction cavities and CRISPR detection cavities pre-stored with corresponding RPA primer pairs and sgRNAs in the reagent combination of claim 1.

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