Rapid detection method for three pathogenic bacteria of rice based on RPA-CRISPR / Cas12b technology

Abstract

The invention discloses a rapid detection method for three pathogenic bacteria of rice 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 for three quarantine pests, namely BG (rice blight), XOC (rice bacterial leaf streak) and XOO (rice bacterial leaf blight). 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 rice 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 rice bacterial brown spot (BG), rice bacterial leaf streak (XOC) and rice bacterial leaf blight (XOO) based on RPA-CRISPR / Cas12b technology. BACKGROUND

[0002] Rice is a major food crop in the world, and its production safety is crucial. However, various quarantine pathogens such as rice bacterial brown spot (BG), rice bacterial leaf streak (XOC) and rice bacterial leaf blight (XOO) seriously harm the growth of rice and cause yield loss or even complete loss. Therefore, developing a rapid, accurate and practical detection technology is of great significance for rice disease control and port quarantine.

[0003] At present, the detection of pathogenic bacteria mainly relies on molecular detection technologies 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 severely dependent on a thermal cycler, is complicated to operate and time-consuming, and is difficult to apply to on-site rapid detection. Isothermal amplification technologies such as LAMP and RPA overcome the dependence on temperature cycling to some extent, significantly shorten the amplification time, and provide the possibility for the development of 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, 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] To achieve the above application purposes, the application adopts the following technical solutions: The application provides a reagent combination for simultaneously detecting multiple pathogenic bacteria of rice, the multiple pathogenic bacteria being rice bacterial brown spot Burkholderia glumae, referred to as BG, rice bacterial leaf streak Xanthomonas oryzaepv. Oryzicola, referred to as XOC, and rice bacterial leaf blight Xanthomonas oryzae pv. Oryzae, referred to as XOO; the reagent combination comprising specific RPA primer pairs and sgRNA. When the pathogenic bacteria is BG, 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; When the pathogenic bacteria is XOC, 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 its nucleotide sequence is as shown in SEQ ID NO. 6; When the pathogenic bacteria is XOO, 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 its nucleotide sequence is as shown in SEQ ID NO. 9.

[0007] The application also provides a kit for simultaneously detecting multiple pathogenic bacteria of rice, 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.

[0008] The application also provides a method for simultaneously detecting multiple pathogenic bacteria of rice 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 rice stem rot bacteria, rice bacterial leaf blight bacteria or rice bacterial leaf blight bacteria according to the fluorescence 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 fluorescence signal detection are all automatically completed on a centrifugal microfluidic chip; the centrifugal microfluidic chip comprises a mutually isolated RPA reaction cavity and CRISPR detection cavity pre-provisioned with corresponding RPA primer pairs and sgRNA in the reagent combination. Advantages

[0011] (1) The RPA isothermal amplification technology adopted in the present 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 result acquisition takes no more than 30 minutes, far exceeding the traditional PCR technology (usually 1.5-2 hours), greatly improving the detection efficiency and meeting the needs of emergency monitoring and on-site rapid decision-making.

[0012] (2) The present application uses a combination of specific RPA primers and sgRNA designed with care, and the double recognition and signal amplification of the target sequence by the CRISPR-Cas12b system, so that the detection method has high sensitivity and specificity for the detection of rice bacterial sheath blight (BG), rice bacterial leaf streak (XOC) and rice bacterial leaf blight (XOO), far superior to conventional PCR methods. And no false positive signal appeared in the cross reaction test, ensuring the accuracy and reliability of the detection results.

[0013] (3) The present application not only provides a detection scheme for a single pathogenic bacterium, but more importantly, through the multi-chamber design of the microfluidic chip, BG, XOC and XOO three pathogenic bacteria can be simultaneously and parallelly detected on one chip, and the screening of multiple potential threat factors can be completed at one time, greatly improving the detection throughput and efficiency.

[0014] (4) The present 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 relying on portable equipment, without the need for complex thermal cycler and professional operators, greatly reducing the use threshold and being suitable for limited resources such as grassroots units, ports and field sites. BRIEF DESCRIPTION OF DRAWINGS

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

[0016] Figure 2 Figure is the performance screening result graph of three candidate sgRNAs of XOC target in the embodiment of the present application; (A) is the fluorescence curve; (B) is the fluorescence change rate.

[0017] Figure 3Figure of performance screening result of three candidate sgRNAs of XOO target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0018] Figure 4 Figure of performance screening result of different RPA primer combinations of BG target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0019] Figure 5 Figure of performance screening result of different RPA primer combinations of XOC target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0020] Figure 6 Figure of performance screening result of different RPA primer combinations of XOO target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0021] Figure 7 Figure of sensitivity verification result of BG target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0022] Figure 8 Figure of sensitivity verification result of XOC target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0023] Figure 9 Figure of sensitivity verification result of XOO target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0024] Figure 10 Figure of specificity verification result of BG target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0025] Figure 11 Figure of specificity verification result of XOC target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0026] Figure 12 Figure of specificity verification result of XOO target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0027] Figure 13 Figure of subtype compatibility verification result of BG target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0028] Figure 14 Figure of subtype compatibility verification result of XOC target in the embodiment of the application; (A) is fluorescence curve; (B) is fluorescence change rate.

[0029] Figure 15It is a XOO target subtype compatibility verification result graph in the embodiment of the application; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0030] Figure 16 It is a BG target genome test rapid extraction process interference verification result graph of the process; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0031] Figure 17 It is a XOC target genome test rapid extraction process interference verification result graph of the process; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0032] Figure 18 It is a XOO target genome test rapid extraction process interference verification result graph of the process; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0033] Figure 19 It is a BG target bacteria liquid different concentration rapid extraction process detection effect graph; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0034] Figure 20 It is a XOC target bacteria liquid different concentration rapid extraction process detection effect graph; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0035] Figure 21 It is a XOO target bacteria liquid different concentration rapid extraction process detection effect graph; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0036] Figure 22 It is a BG target non-rapid extraction system bacteria liquid lysis test result graph; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0037] Figure 23 It is a XOC target non-rapid extraction system bacteria liquid lysis test result graph; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0038] Figure 24 It is a XOO target non-rapid extraction system bacteria liquid lysis test result graph; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0039] Figure 25 It is a BG target bacteria liquid rapid extraction process sensitivity verification result graph; (A) is a fluorescence curve; (B) is a fluorescence rate of change.

[0040] Figure 26It is a result chart of sensitivity verification of the XOC target bacteria liquid rapid extraction process in the embodiment of the application; (A) is a fluorescence curve; (B) is a fluorescence change rate.

[0041] Figure 27 It is a result chart of sensitivity verification of the XOO target bacteria liquid rapid extraction process in the embodiment of the application; (A) is a fluorescence curve; (B) is a fluorescence change rate.

[0042] Figure 28 It is an effect chart of the chip primary detection BG, XOC and XOO target nucleic acid in the embodiment of the application.

[0043] Figure 29 It is an effect chart of the chip test BG target different primer pairs in the embodiment of the application.

[0044] Figure 30 It is a detection effect chart of the chip confirming BG, XOC and XOO targets in the embodiment of the application.

[0045] Figure 31 It is a result chart of the BG target bacteria liquid chip sensitivity verification in the embodiment of the application.

[0046] Figure 32 It is a result chart of the XOC target bacteria liquid chip sensitivity verification in the embodiment of the application.

[0047] Figure 33 It is a result chart of the XOO target bacteria liquid chip sensitivity verification in the embodiment of the application. DETAILED DESCRIPTION

[0048] In order to enable personnel in the art to better understand the technical solutions in the application, the following will further describe the application in detail with reference to the embodiments.

[0049] The nucleotide sequence involved in the application is as follows: SEQ ID NO. 1: the sequence shown is the nucleotide sequence of the RPA primer RPA-BG-F3.

[0050] SEQ ID NO. 2: the sequence shown is the nucleotide sequence of the RPA primer RPA-BG-R4.

[0051] SEQ ID NO. 3: the sequence shown is the nucleotide sequence of the sgRNA target BG-sgRNA.

[0052] SEQ ID NO. 4: the sequence shown is the nucleotide sequence of the RPA primer RPA-XOC-F3.

[0053] SEQ ID NO. 5: the sequence shown is the nucleotide sequence of the RPA primer RPA-XOC-R2.

[0054] SEQ ID NO. 6: The sequence shown is the nucleotide sequence of sgRNA target XOC-sgRNA-2.

[0055] SEQ ID NO. 7: The sequence shown is the nucleotide sequence of RPA primer RPA-XOO-F3.

[0056] SEQ ID NO. 8: The sequence shown is the nucleotide sequence of RPA primer RPA-XOO-R1.

[0057] SEQ ID NO. 9: The sequence shown is the nucleotide sequence of sgRNA target XOO-sgRNA.

[0058] SEQ ID NO. 10: The sequence shown is the nucleotide sequence of sgRNA target Bg-ITS-2.

[0059] SEQ ID NO. 11 : The sequence shown is the nucleotide sequence of sgRNA target Bg-GyrB-1.

[0060] SEQ ID NO. 12: The sequence shown is the nucleotide sequence of sgRNA target XOO-tale-2.

[0061] SEQ ID NO. 13: The sequence shown is the nucleotide sequence of sgRNA target XOO-rhs-1.

[0062] SEQ ID NO. 14: The sequence shown is the nucleotide sequence of sgRNA target XOC-sgRNA-1.

[0063] SEQ ID NO. 15: The sequence shown is the nucleotide sequence of sgRNA target XOC-sgRNA-3.

[0064] SEQ ID NO. 16: The sequence shown is the nucleotide sequence of PCR primer Bg-F.

[0065] SEQ ID NO. 17: The sequence shown is the nucleotide sequence of PCR primer Bg-R.

[0066] SEQ ID NO. 18: The sequence shown is the nucleotide sequence of PCR primer Bg_G_B_gyrBF1.

[0067] SEQ ID NO. 19: The sequence shown is the nucleotide sequence of PCR primer Bg_G_B_gyrBR1.

[0068] SEQ ID NO. 20: The sequence shown is the nucleotide sequence of PCR primer Bg-impEF1.

[0069] SEQ ID NO. 21: The sequence shown is the nucleotide sequence of PCR primer Bg- impER1.

[0070] SEQ ID NO. 22: The sequence shown is the nucleotide sequence of PCR primer Xoo-F.

[0071] SEQ ID NO. 23: The sequence shown is the nucleotide sequence of PCR primer Xoo-R.

[0072] SEQ ID NO. 24: The sequence shown is the nucleotide sequence of PCR primer Xoo_rhsF1.

[0073] SEQ ID NO. 25: The sequence shown is the nucleotide sequence of PCR primer Xoo_rhsR1.

[0074] SEQ ID NO. 26: The sequence shown is the nucleotide sequence of PCR primer Xoo_avrF1.

[0075] SEQ ID NO. 27: The sequence shown is the nucleotide sequence of PCR primer Xoo_avrR1.

[0076] SEQ ID NO. 28: The sequence shown is the nucleotide sequence of PCR primer XOC-F.

[0077] SEQ ID NO. 29: The sequence shown is the nucleotide sequence of PCR primer XOC-R.

[0078] SEQ ID NO. 30: The sequence shown is the nucleotide sequence of RPA primer RPA- BG-F1.

[0079] SEQ ID NO. 31: The sequence shown is the nucleotide sequence of RPA primer RPA- BG-F2.

[0080] SEQ ID NO. 32: The sequence shown is the nucleotide sequence of RPA primer RPA- BG-R1.

[0081] SEQ ID NO. 33: The sequence shown is the nucleotide sequence of RPA primer RPA- BG-R2.

[0082] SEQ ID NO. 34: The sequence shown is the nucleotide sequence of RPA primer RPA- BG-R3.

[0083] SEQ ID NO. 35: The sequence shown is the nucleotide sequence of RPA primer RPA- XOC-F1.

[0084] SEQ ID NO.36: The sequence shown is the nucleotide sequence of RPA primer RPA-XOC-R1.

[0085] SEQ ID NO.37: The sequence shown is the nucleotide sequence of the RPA primer RPA-XOC-F2.

[0086] SEQ ID NO.38: The sequence shown is the nucleotide sequence of the RPA primer RPA-XOC-R3.

[0087] SEQ ID NO.39: The sequence shown is the nucleotide sequence of RPA primer XOO-RPA-F1.

[0088] SEQ ID NO.40: The sequence shown is the nucleotide sequence of RPA primer XOO-RPA-F2.

[0089] SEQ ID NO.41: The sequence shown is the nucleotide sequence of RPA primer XOO-RPA-R2. Example

[0090] 1. Experimental materials 1.1 Instruments and Equipment SynSor Portable Multi-Target Rapid Detection Device, XS-D-001 – Xunshi Biotechnology; QPCR Instrument - SLAN-96P – Shanghai Hongshi Medical Technology Co., Ltd.; PCR Instrument (TC-S / 96 / G / H(b)BA) – Hangzhou Bori Technology Co., Ltd.; Qubit 3.0 Fluorometer – Thermo Fisher Scientific; Nanodrop 2000 Ultra-Micro Spectrophotometer – Thermo Fisher Scientific; Metal Bath (DH100-2) – Hangzhou Ruicheng Instrument Co., Ltd.

[0091] 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.

[0092] 2. Experimental Methods 2.1, Detection target Target design development was performed for the target regions in Table 1.

[0093]

[0094] Actual sample performance verification was performed using 19 nucleic acid samples and 3 bacterial liquid samples, and in addition, a bacterial liquid for each target was tested, and the corresponding sample information is shown in Table 2 below.

[0095]

[0096] 2.2, sgRNA design and verification 2.2.1, sgRNA design According to the selected region or the commonly used target region in the literature, combined with the host background genome that needs to be avoided, the sgRNA was designed and scored according to the PAM position, GC%, internal dimer structure, fragment structure openness, base position preference, specificity, etc. The score can be understood as the success probability. Generally, 3 candidate fragments with a score of 40 or more are retained. If the score is generally lower than 40, the number of candidate fragments needs to be increased to improve the success probability. If the sequence in the spacer has the risk of destroying the backbone secondary structure, it will be directly excluded from the candidate list to avoid the risk of specificity and sensitivity.

[0097] Based on the above design principles, sgRNA was 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 base pairs matched across species was <15 nt, and the specificity was good. The sgRNA used is shown in Table 3.

[0098]

[0099] 2.2.2, sgRNA performance verification 2.2.2.1, PCR primer design According to the position of the sgRNA design, the region within 100 bp upstream and downstream of the sgRNA was selected to design the PCR primer (see Table 4). The primer was designed using commonly used software for designing PCR primers, 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 are verified for performance under the same template.

[0100]

[0101] 2.2.2.2, Template amplification verification PCR amplification was performed using plasmid / template DNA and corresponding primers 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.

[0102]

[0103]

[0104] 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, and the concentration of the target to be tested should be higher than 2 ng / μL, and there was obvious distinction with the concentration of the blank control.

[0105] 2.2.2.3, sgRNA verification The CRISPR system was prepared according to the system in Table 7.

[0106]

[0107] 1 μL of about 10~100 ng of PCR amplification product was mixed with the above CRISPR system, and the qPCR instrument was set at 43℃ for 15 min, and the FAM fluorescence signal was collected every minute. According to the curve change of fluorescence signal, the performance or specificity of sgRNA was preliminarily verified. The endpoint of the fluorescence value curve of the target amplification product and the negative control should be significantly different.

[0108] The calculation formula of fluorescence intensity growth rate is as follows: .

[0109] Wherein, Fluorescence Slope represents the fluorescence growth rate, Rn represents the fluorescence signal of the nth minute, and R1 represents the fluorescence signal of the first minute. Fluorescence Slope greater than 0.5 is determined as positive, and the Fluorescence Slope of the negative group should not exceed 0.2.

[0110] 2.3, primer design and verification 2.3.1, RPA primer design In the detection range of the target, the fragments of 30-35 base length were selected as the RPA primer candidates; in order to ensure stability and specificity, the GC content of the RPA primer should be between 40%-60%, and the RPA amplification fragment was usually 100-200 bp. Based on this primer design principle, 3 upstream and downstream primers were designed for RPA primer verification, and NCBI primer-BLAST was used for amplification coverage and specificity verification.

[0111] Based on the above design principles, the RPA primers shown in Table 8 were designed for each plasmid / template for subsequent synthesis and verification experiments.

[0112]

[0113] 2.4, Chip verification 2.4.1, Chip detection process Nucleic acids and bacterial liquid were used for verification in this part.

[0114] Using the system determined in 3.2, the verification of the target nucleic acid sample was carried out. First, the sample was quickly extracted, and then the sample lysate was diluted 8 times to prepare the RPA system. After mixing evenly, the chip was detected, and the fluorescence signal was judged according to the shooting.

[0115] The test process of the chip is shown in Table 9, and the total detection time is about 28 min.

[0116]

[0117] 3, Experimental results 3.1, Screening results of sgRNA The fluorescence intensity of the amplification products of each sgRNA and the corresponding template was determined by CRISPR reaction. The amplification curve and fluorescence growth rate analysis results are shown in Figures 1-3 , and the results of fluorescence value change are shown in Table 10. The analysis results show that: BG target Bg-sgRNA (SEQ ID NO. 3), XOC target XOC-sgRNA-2 (SEQ ID NO. 6) and XOO target XOO-sgRNA (SEQ ID NO. 9) have better effects.

[0118]

[0119] 3.2, Screening results of RPA primers The nucleic acid sample was diluted to 10 ng / μL to screen different combinations of RPA primers, and two-step CRISPR trans reaction was used. The first step was RPA amplification reaction, and then the RPA amplification product was mixed with CRISPR reaction solution for CRISPR reaction.

[0120] 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.

[0121] The effective amplification rates of each primer at each dilution gradient are shown in Tables 11, 12, and 13.

[0122]

[0123]

[0124]

[0125] The analysis results show that: (1) All primers designed for the BG target were able to amplify the sample. After comparing their performance, the RPA-BG-F3 / R4 (SEQ ID NO.1 / SEQ ID NO.2) primer pair with better amplification performance was selected. Figure 4 Proceed to the next step of sensitivity verification.

[0126] (2) Most of the primers designed for the XOC target were able to amplify the sample. After comparing their performance, the RPA-XOC-F3 / R2 (SEQ ID NO.4 / SEQ ID NO.5) primer pair with better amplification performance was selected. Figure 5 Proceed to the next step of sensitivity verification.

[0127] (3) Most of the primers designed for the XOO target were able to amplify the sample. After comparing the performance, the RPA-XOO-F3 / R1 (SEQ ID NO.7 / SEQ ID NO.8) primer pair with better amplification performance was selected. Figure 6 Proceed to the next step of sensitivity verification.

[0128] 3.3 Construction and Validation of the Testing System 3.3.1 Validation of the sensitivity of the detection system A serial dilution was performed using the genomic template, with the genomic addition levels per unit reaction set at 156 pg, 15.6 pg, 7.8 pg, 1.56 pg, 0.78 pg, 0.156 pg, and 0.078 pg, adjusted appropriately based on the effects on different targets. The primer and sgRNA combinations selected in section 3.2, along with the same CRISPR system, were used to validate the sensitivity of the target. Each dilution was repeated three times for validation.

[0129] The amplification curves and fluorescence signal growth rates for sensitivity verification of each target are shown below, BG (Figure 7 XOC (Xanthomonas oryzae), XOO (Xanthomonas oryzae pv. oryzae), BG (Bacillus Figure 8 XOC (Xanthomonas oryzae), XOO (Xanthomonas oryzae pv. oryzae), BG (Bacillus Figure 9 The three targets can detect at least 0.156 pg of nucleic acid sample.

[0130] According to the significance analysis of the sensitivity verification experiment results, the detection limit of each primer and corresponding target can be preliminarily judged as shown in Table 14.

[0131]

[0132] According to the relationship between the genome copy number and the genome quality in the previous study, the detection limit of XOC is about 31 copies / Test, the sensitivity of XOO is about 2.2 copies / Test, and the sensitivity of BG is about 5.7 copies / Test.

[0133] 3.3.2, detection system specificity verification Five 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.

[0134] The results of the specificity verification of the BG target are shown in the following figure: Figure 10 The verification results show that the BG target detects BG samples as positive, and the detection results of the five cross-species are negative, indicating that the BG target has good specificity in cross-species.

[0135] The results of the specificity verification of the XOC target are shown in the following figure: Figure 11 The verification results show that the XOC target detects XOC samples as positive, and the detection results of Xa, Xap, Xam, Xav, Xcm in the five cross-species are negative, indicating that the XOC target has good specificity in cross-species.

[0136] The results of the specificity verification of the XOO target are shown in the following figure: Figure 12 The verification results show that the XOO target detects XOO samples as positive, and the detection results of Xa, Xap, Xam, Xav, Xcm in the five cross-species are negative, indicating that the XOO target has good specificity in cross-species.

[0137] 3.3.3, detection system sample compatibility test The nucleic acid was diluted to 10 ng / μL for this part of the verification.

[0138] Using the existing detection system, the actual sample of the target nucleic acid was verified. Each actual sample was verified twice.

[0139] The amplification curves and fluorescence signal growth rates of different subtype genomic samples are shown below. BG targets can be compatible with BG2, BG3 subtypes ( Figure 13 ); XOC targets can be compatible with XOC2, XOC3 subtypes ( Figure 14 ); XOO targets can be compatible with XOO3, and cannot be compatible with XOO2 ( Figure 15 ).

[0140] 3.4, Development of rapid extraction process of bacterial liquid sample 3.4.1, Evaluation of the interference of rapid extraction process on the system The interference of the rapid extraction process on the detection system was tested using three target genes. The mixed solution obtained from the original configuration sample (hereinafter referred to as the original solution) was used as a positive control, and the rapid extraction process sample was used as a test group. The primers and sgRNA combinations determined in 3.2 were used for verification, and the same CRISPR system was used for verification. Each cross-reaction was verified twice.

[0141] The verification results of BG target ( Figure 16 ), XOC target ( Figure 17 ), and XOO target ( Figure 18 ) show that the original solution is positive, and the detection effect of the rapid extraction sample is similar to that of the original solution, without obvious reduction. Moreover, the three targets have the same trend, indicating that the rapid extraction process has no obvious interference on the detection system.

[0142] 3.4.2, Feasibility verification of bacterial liquid rapid extraction system The bacterial liquid was diluted 10 times and 100 times for this part of the verification.

[0143] Using the existing detection system, the target target bacterial liquid sample was verified. Each actual sample was verified twice.

[0144] The amplification curves and fluorescence signal growth rates of different concentrations of bacterial liquid samples are shown below. BG target two concentration bacterial liquid samples can be detected ( Figure 19 ); XOC target two concentration bacterial liquid samples can be detected ( Figure 20 ); XOO bacterial liquid two concentrations cannot be detected ( Figure 21 ). Overall, the rapid extraction process can be used to detect bacterial liquid.

[0145] 3.4.3, Performance verification of non-rapid extraction system bacterial liquid Test with 1E6 CFU / mL of bacterial liquid sample. Use the primer, template and sgRNA combination determined in 3.2, and the same CRISPR system. Use the rapid extraction system as a positive control, and test the bacterial liquid lysis effect of the non-rapid extraction process system (normal saline). Each test is repeated twice for verification.

[0146] The detection effect of each target normal saline lysis bacterial liquid is shown below, BG target ( Figure 22 ), XOC target ( Figure 23 ) cannot be detected with normal saline lysis, and the effect is obviously different from that of the rapid extraction process system, further illustrating that the rapid extraction process has obvious nucleic acid release effect on bacterial liquid. The rapid extraction and normal saline lysis of XOO target bacterial liquid do not produce positive signals, which is the same as 3.4.2, and is a manifestation of subtype incompatibility. See Figure 24 .

[0147] 3.4.4, sensitivity verification of bacterial liquid rapid extraction process Dilute the bacterial liquid sample by gradient dilution. The gradient settings include 1E6 CFU / mL, 1E5 CFU / mL, 1E4 CFU / mL, 1E3 CFU / mL, 1E2 CFU / mL, 1E1 CFU / mL, 1 CFU / mL, corresponding to 156 CFU, 15.6 CFU, 1.56 CFU, 0.156 CFU, 0.0156 CFU, 0.00156 CFU, 0.000156 CFU of single reaction bacterial number, and adjust appropriately according to the effect of different targets. Use the primer, template and sgRNA combination determined in 3.2, and the same CRISPR system to verify the sensitivity of the target bacterial liquid. Each gradient is repeated twice for verification.

[0148] The amplification curves and fluorescence signal growth rates of the sensitivity verification of each target bacterial liquid are shown below. The BG target can detect 15.6 CFU / T bacterial liquid sample at the lowest ( Figure 25 ); the XOC target can detect 1.56 CFU / T bacterial liquid sample at the lowest ( Figure 26 ); and the XOO target bacterial liquid sample is not detected ( Figure 27 ).

[0149] 3.5, chip detection results 3.5.1, initial chip detection results Dilute the nucleic acid sample to a concentration of 10 ng / μL. First, use the rapid extraction process to test on the chip, and the verification results of each target are as follows Figure 28 . The BG target genome is not detected, the XOC target genome can be detected, the XOO2 target is not detected, and the XOO3 target can be detected.

[0150] 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 with good effect screened in 3.2: RPA-BG-F1 / R3, RPA-BG-F1 / R4, RPA-BG-F2 / R4, RPA-BG-F3 / R4 were subjected to chip test, and two-step CRISPR reaction was used. The first step was RPA amplification reaction, and then the RPA amplification product was mixed with CRISPR reaction solution for CRISPR reaction. The positive and negative results were judged according to the fluorescence signal.

[0151] The verification results of different primer pairs of BG target are as follows Figure 29 RPA-BG-F1 / R3, RPA-BG-F2 / R4, RPA-BG-F3 / R4 were not detected, and RPA-BG-F1 / R4 could detect positive signals.

[0152] 3.5.3, Chip confirmation BG, XOC, XOO three target detection effect The BG, XOC, XOO target nucleic acid samples were diluted to 10 ng / μL, and the target primers and sgRNA screened in 3.2 were used for verification, wherein the BG primer pair was replaced with RPA-BG-F1 / R4 for chip test. Two-step CRISPR reverse reaction was used. The first step was RPA amplification reaction, and then the RPA amplification product was mixed with CRISPR reaction solution for CRISPR reaction. After completing the chip process, the results were judged according to the brightness change of the detection hole. In the chip setting, hole 1 corresponds to BG target, hole 2 corresponds to XOC target, and hole 3 corresponds to XOO target. Each chip adds one target sample.

[0153] The verification results of the three targets are as follows Figure 30 . The BG target detection system corresponds to hole 1 on three chips. It can be seen that only when the sample is BG sample, it is positive, and when the sample is XOC and XOO, it is negative; the XOC target detection system corresponds to chip hole 2, only when the sample is XOC genome, there is a positive signal, and when the sample is BG and XOO, it is negative; the XOO target detection system corresponds to chip hole 3, when the sample is XOO genome, it is positive, and when the sample is BG and XOC, it is negative.

[0154] 3.5.4, Sensitivity of three target bacteria liquid rapid extraction under chip process Gradient dilution was performed with bacterial liquid or genomic sample, and the target primer screened in 3.2 was verified by sgRNA. The two-step CRISPR transreaction was adopted, the first step was RPA amplification reaction, 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 the positive and negative (the positive well has a square box outside the well).

[0155] The BG target bacterial liquid chip detection result is as shown in Figure 31 The 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 bacterial liquid sample that can be detected is 15.6 CFU.

[0156] The XOC target bacterial liquid chip detection result is as shown in Figure 32 The XOC target can be detected after rapid extraction, the whole process is completed, and the minimum bacterial liquid sample that can be detected is 15.6 CFU.

[0157] The XOO target genomic chip detection result is as shown in Figure 33 The XOO target chip can be detected, and the minimum genomic sample that can be detected in a single well under the chip process is 1 pg / T, which is equivalent to about 22 copies that can be detected per reaction.

[0158] In summary, the present application develops an isothermal amplification detection method based on RPA and CRISPR for BG, XOC and XOO targets, which breaks away from the dependence on complex instrument equipment platform of traditional molecular biology methods and greatly simplifies 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 detection technology of the target is verified in terms of sensitivity, specificity and POCT platform. 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: (1) The BG target sgRNA is Bg-sgRNA, which can be tested with primers RPA-BG-F3 / R4. The minimum detectable genome is 0.156 pg per reaction, which is equivalent to about 5.7 copies that can be detected per reaction, and is specific in BG-CK1-BG-CK5. The minimum detectable bacterial liquid is 15.6 CFU / T under the rapid extraction process and detection system, and the minimum detectable bacterial liquid on the chip is 15.6 CFU / T.

[0159] (2) The XOC target sgRNA XOC-sgRNA-2 has good binding with the primers RPA-XOC-F3 / R2, and the minimum detection genome is 0.156 pg per reaction, which is equivalent to 31 copies per reaction, and is specific in the five cross-species Xa, Xap, Xam, Xav and Xcm, and can contain two subtypes XOC2 and XOC3. The minimum detectable bacteria liquid is 1.56 CFU / T using the rapid extraction process combined with the detection system. The minimum detectable bacteria liquid on the chip is 15.6 CFU / T.

[0160] (3) The XOO target sgRNA XOO-sgRNA has good detection effect when combined with RPA-XOO-F3 / R1, with a minimum of 0.156 pg per reaction, which is equivalent to 2.2 copies per reaction, and is specific in the five cross-species Xa, Xap, Xam, Xav and Xcm, and cannot contain the XOO2 subtype, but can contain the XOO3 subtype. The minimum detectable genomic sample is 1 pg / T on the chip combined with the rapid extraction process, which is equivalent to 14.1 copies per reaction.

Claims

1. A reagent combination for simultaneously detecting a plurality of pathogenic bacteria of rice, characterized by comprising: The multiple pathogenic bacteria are Burkholderia glumae (BG), Xanthomonas oryzae pv. Oryzicola (XOC) and Xanthomonas oryzae pv. Oryzae (XOO); the reagent combination comprises specific RPA primer pairs and sgRNA; When the pathogenic bacteria is BG, 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; When the pathogenic bacteria is XOC, 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 its nucleotide sequence is as shown in SEQ ID NO. 6; When the pathogenic bacteria is XOO, 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 its nucleotide sequence is as shown in SEQ ID NO.

9.

2. A reagent kit for simultaneously detecting multiple pathogens in rice, 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 simultaneously detecting a plurality of pathogenic bacteria of rice for non-diagnostic purposes, characterized by, Comprise the following steps: S1, using the nucleic acid of the sample to be tested as a template, using the corresponding specific RPA primer pair in the reagent combination of claim 1 to perform RPA reaction; 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 determining whether the sample to be tested contains Burkholderia glumae, Xanthomonas oryzae pv. Oryzicola or Xanthomonas oryzae pv. Oryzae 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 RPA reaction cavities and CRISPR detection cavities which are mutually isolated and preloaded with the corresponding RPA primer pairs and sgRNA in the reagent combination of claim 1.

Images