CN116656850B - Sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof - Google Patents

Sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof Download PDF

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CN116656850B
CN116656850B CN202310926755.3A CN202310926755A CN116656850B CN 116656850 B CN116656850 B CN 116656850B CN 202310926755 A CN202310926755 A CN 202310926755A CN 116656850 B CN116656850 B CN 116656850B
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赵辉
张海敏
胡帅
张金鑫
纪长绵
杜晓希
贾瑞宗
张雨良
郭安平
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Sanya Research Institute Chinese Academy Of Tropical Agricultural Sciences
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Abstract

The invention provides a sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof, belonging to the technical field of pathogenic microorganism detection. The invention provides an RPA primer pair and specific crRNA for detecting rice bacterial leaf blight bacteria. The invention also provides a kit and a method for rapidly detecting rice bacterial blight bacteria based on CRISPR/Cas12 a-RPA. The detection method combines the RPA amplification method with the CRISPR/Cas12a detection method to detect the bacterial blight of rice, and realizes visual detection through a blue light gel cutting instrument and a lateral flow chromatography test strip. The nucleic acid detection method is simple and quick, has good specificity and sensitivity, can detect by only needing a simple constant temperature heater, and has the advantage of quick nucleic acid detection in the field and the field.

Description

Sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof
Technical Field
The invention relates to the technical field of pathogenic microorganism detection, in particular to a method and a kit for rapidly detecting bacterial leaf blight of rice based on CRISPR/Cas12 a-RPA.
Background
Bacterial leaf blight of rice is a bacterial disease caused by xanthomonas oryzae, which severely damages rice yield. Bacterial leaf blight of rice is reported in all continents (except europe), and in extreme cases, millions of hectares of rice are affected annually, with losses as high as 75%. Before obvious symptoms appear, rice infected with bacterial leaf blight of rice in early stage is difficult to carry out physical diagnosis through nonprofessional knowledge, so that outbreaks of bacterial leaf blight of rice cause massive death of rice crops. Therefore, the two diseases are detected in the rice seed bacteria-carrying stage or the rice seedling stage through a molecular technology, and the method has important significance for increasing income of farmers in rice production.
At present, the method for detecting the bacterial leaf blight of rice mainly comprises a traditional detection method, a molecular detection technology developed in recent years and the like. The traditional detection method mainly comprises phage detection method, seedling growth observation method, pathogenicity determination, direct separation method, serological detection technology and the like; the molecular detection technology developed in recent years mainly comprises the following steps: common PCR, digital PCR, fluorescent quantitative PCR, and the like. The traditional detection method is long in time consumption, inconvenient to detect, needs special technicians to detect, has specialization like phage detection method, is easy to cause false negative, and serological detection technology is easy to cause false positive. In recent years, the development of molecular biotechnology such as PCR technology and fluorescent quantitative PCR technology requires specialized personnel, expensive experimental instruments and specific experimental sites, and has the disadvantages of long time consumption and inconvenient detection; at present, national standard for detecting bacterial leaf blight of rice in China is based on detection of iron-containing cell receptor factor genes by utilizing PCR technology.
CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-related) is a naturally occurring adaptive immune system in prokaryotes such as bacteria and archaea, which has been widely used for the diagnosis of various infectious microorganisms, including viruses and bacteria, as Cas proteins with different activities are continuously discovered. In addition, the system can be used for detecting microRNAs (miRNAs), single Nucleotide Polymorphisms (SNPs), DNA methylation and the like. Cas12a enzymes in class II CRISPR proteins, in addition to having specific targeting, also exhibit the ability to cleave non-specifically from neighboring nucleic acids, have been widely used in conjunction with isothermal amplification techniques for the diagnosis of various microorganisms. The method can be used for rapidly detecting and diagnosing the bacterial leaf blight of the rice in the field due to strong specificity, high sensitivity, convenience and rapidness.
Disclosure of Invention
Aiming at the problems, the invention provides a method and a kit for rapidly detecting bacterial blight of rice based on CRISPR/Cas12a technology combined with RPA technology, which have the characteristics of good specificity, high sensitivity, simplicity, rapidness, low sample requirement, high efficiency and the like, can realize the requirement of rapid detection in fields, improve the detection efficiency, and can provide a new choice for the detection method of bacterial blight of rice.
In order to achieve the above purpose, the technical scheme of the invention is as follows: a sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA comprises an RPA primer sequence, a crRNA sequence and a ssDNA probe sequence designed according to GSFP genes,
the RPA primer sequence is as follows:
the forward primer RPA-GSFP-F1 is: 5'-TTGGCTAATGACATGGAAATGGTTCCCGTG-3', (SEQ No. 1),
the reverse primer RPA-GSFP-R1 is: 5'-AATTCAAGCTCCGATGCGAAATAGGCACGC-3', (SEQ No. 2),
the crRNA sequence is: UAAUUUCUACUAAGUGUAGAUACUGUAGACAUCGCAGAUCAACA, (SEQ No. 3),
the ssDNA probe sequence comprises any one of ssDNA probe sequences of rice bacterial leaf blight bacteria for fluorescent quantitative PCR instrument detection, ssDNA probe sequences of rice bacterial leaf blight bacteria for visual fluorescent detection and ssDNA probe sequences of rice bacterial leaf blight bacteria for lateral flow chromatography test strip detection.
Further, the ssDNA probe sequence of the rice bacterial leaf blight bacteria for fluorescent quantitative PCR instrument detection is as follows:
ssDNA-reporter-FAM is: 5' -6' FAM-TTATT-BHQ1-3',
dsDNA-reporter-CY5 is: 5'-CY5-TGTCTTATcccccATAAGACA-BHQ1-3'.
Further, the ssDNA probe sequence for visual fluorescence detection of rice bacterial leaf blight bacteria is as follows:
ssDNA-reporter-FAM-2 is: 5' -6' FAM-TGTCTTATcccccATAAGACA-BHQ1-3'.
Further, the ssDNA probe sequence of the rice bacterial leaf blight bacteria for detecting the lateral flow chromatography test strip is as follows: the FB-reporter is: 5' -6' FAM-TTTTTTTTTTT-Biotin-3 '.
A detection kit for rapidly detecting bacterial leaf blight of rice comprises the RPA primer sequence, crRNA sequence, ssDNA probe sequence, rehydration Buffer, mgOAC, lyophilized enzyme powder, cas12a enzyme and ddH 2 O. The concentration of the Cas12a enzyme is 50nM, the working concentration of crRNA is 500nM, and the working concentration of the ssDNA probe sequence is 5nM (test strip detection) or 1000nM (fluorescent quantitative PCR instrument detection or blue-light gel cutting instrument detection).
Further, the kit comprises eight rows of PCR reaction tubes with a plurality of independent units, wherein the PCR reaction tubes contain freeze-dried enzyme powder;
the kit is divided into two reaction systems, including a reaction system for performing an RPA reaction and a reaction system for performing CRISPR/Cas12 a;
when the reaction system for carrying out the RPA reaction works, the total volume of the reaction system of 50. Mu.L is: 2.4 mu.L of forward primer RPA-GSFP-F1 at a concentration of 10. Mu.M, 2.4. Mu.L of reverse primer RPA-GSFP-R1 at a concentration of 10. Mu.M, 29.5. Mu.L of reaction buffer、11.2 μL ddH 2 O and 2 mu L of template DNA are mixed uniformly and then are rapidly put into a reaction small tube containing freeze-dried enzyme powder, and after the freeze-dried enzyme powder is dissolved, 2.5 mu L of MgOAC is added finally;
when the reaction system for performing CRISPR/Cas12a is in operation, the reaction system is selected from any one of the following:
the total volume of the reaction system was 20. Mu.L: 2. mu.L SF buffer (Tris-OAC, mgOAC, BSA and DTT), 1. Mu.L crRNA sequence at a concentration of 10. Mu.M, 1. Mu.L RNase inhibitor with an enzyme activity of 40U/. Mu.L, 2. Mu.L ssDNA-reporter-CY5 at a concentration of 10. Mu.M, 1. Mu.L LbCAs12a at a concentration of 1. Mu.M, 2. Mu.L RPA reaction product, 11. Mu.L DEPC treated water;
or a total volume of 20. Mu.L: 2. mu.L SF buffer (Tris-OAC, mgOAC, BSA and DTT), 1. Mu.L crRNA sequence at a concentration of 10. Mu.M, 1. Mu.L enzyme inhibitor with RNA enzyme activity of 40U/. Mu.L, 2. Mu.L ssDNA-reporter-FAM-2 at a concentration of 10. Mu.M, 1. Mu.L LbCAs12a at a concentration of 1. Mu.M, 2. Mu.L RPA reaction product, 11. Mu.L DEPC treated water;
or a total volume of 20. Mu.L: 2. mu.L SF buffer (Tris-OAC, mgOAC, BSA and DTT), 1. Mu.L crRNA at a concentration of 10. Mu.M, 1. Mu.L RNase inhibitor at an enzyme activity of 40U/. Mu.L, 2.5. Mu.L FB-reporter at a concentration of 100 nm, 1. Mu.L LbCAs12a at a concentration of 1. Mu.M, 2. Mu.L RPA product, 10.5. Mu.L DEPC treated water.
The detection kit for rapidly detecting the bacterial leaf blight of the rice is applied to detection of the bacterial leaf blight of the rice in the field.
A detection method for rapidly detecting bacterial leaf blight of rice comprises the following steps:
1) Extracting genome DNA of a rice bacterial leaf blight suspected sample; (a field rapid extraction method of bacterial leaf blight bacteria of rice),
2) Taking the genome DNA extracted in the step 1) as a template, and carrying out RPA amplification reaction by using the kit;
3) Adding the RPA amplification product obtained in the step 2) into a CRISPR/Cas12a reaction system to perform fluorescence detection reaction or RPA/Cas12a-LFA reaction.
Further, the method for extracting genomic DNA of the rice bacterial leaf blight suspected sample in the step 1) comprises the following steps: cutting the leaves of a suspected sample of bacterial leaf blight of rice into fine fragments, placing the fragments in a clean 1.5 mL sterile centrifuge tube, adding 0.5M NaOH solution, mashing the leaves with a sterilized grinding rod, standing for 1 min at room temperature, and diluting by using TE Buffer (Tris-EDTA) for 50 times;
in step 2), 50. Mu.L of the reaction system was: 2.4 mu.L of forward primer RPA-GSFP-F1 at a concentration of 10. Mu.M, 2.4. Mu.L of reverse primer RPA-GSFP-R1 at a concentration of 10. Mu.M, 29.5. Mu. L Rehydration Buffer, 11.2. Mu.L of ddH 2 O and 2 mu L of template DNA are mixed uniformly and then are quickly placed into a reaction small tube containing freeze-dried enzyme powder, 2.5 mu L of MgOAC is added on a tube cover after the freeze-dried enzyme powder is dissolved, and the reaction conditions are as follows: amplifying for 10 min at 39 ℃;
in step 3), 20. Mu.L of the reaction system was: 2. mu.L SF buffer, 1. Mu.L crRNA sequence with concentration of 10. Mu.M, 1. Mu.L RNase inhibitor with enzyme activity of 40U/. Mu.L, 2. Mu.L ssDNA-reporter-CY5 with concentration of 10. Mu.M, 1. Mu.L LbCAs12a with concentration of 1. Mu.M, 2. Mu.L RPA reaction product, 11. Mu.L DEPC treated water, and the reaction is carried out in a real-time fluorescence quantitative PCR instrument at 37 ℃ for reaction of 1 h;
or 20. Mu.L of the reaction system: 2. mu.L SF buffer, 1. Mu.L crRNA sequence at a concentration of 10. Mu.M, 1. Mu.L RNase inhibitor with an enzyme activity of 40U/. Mu.L, 2. Mu.L ssDNA-reporter-FAM-2 at a concentration of 10. Mu.M, 1. Mu.L LbCAs12a at a concentration of 1. Mu.M, 2. Mu.L RPA reaction product, 11. Mu.L DEPC treated water under the following conditions: reacting for 5min at 37 ℃, and observing a fluorescence result by a 470 nm blue light gel cutting instrument;
or 20. Mu.L of the reaction system: 2. mu.L SF buffer, 1 mu.L crRNA sequence with concentration of 10 mu M, 1 mu.L RNase inhibitor with enzyme activity of 40U/. Mu.L, 2.5 mu.L FB-reporter with concentration of 100 nm, 1 mu.L LbCAs12a with concentration of 1 mu M, 2 mu.L RPA reaction product, 10.5 mu.L DEPC treated water, reacting for 20 min at 37 ℃, supplementing to 50 mu.L with DEPC treated water, mixing uniformly, dripping the mixture into the binding pad end of the test strip, and observing the color of a detection line (T line) and a quality control line (C line) on the test strip.
The detection method for rapidly detecting the bacterial leaf blight of the rice is applied to the detection of the bacterial leaf blight of the rice.
The sequence combination, the kit and the detection method for rapidly detecting the bacterial leaf blight of the rice based on CRISPR/Cas12a-RPA have the beneficial effects that:
the invention provides a detection kit for rapidly detecting bacterial leaf blight of rice, and also provides a detection method for rapidly detecting bacterial leaf blight of rice. The detection method combines the RPA amplification method with the CRISPR/Cas12 detection method, has good specificity and sensitivity, does not need expensive instruments and equipment, is simple, convenient and quick, has the advantage of quick detection, is more beneficial to popularization and application of molecular detection technology of rice bacterial leaf blight in farmlands, and can effectively monitor, prevent and control the rice bacterial leaf blight.
Drawings
The invention will be described in further detail with reference to the drawings and the detailed description.
FIG. 1 is a schematic diagram of a detection operation flow for detecting bacterial blight of rice based on RPA/Cas12 a;
FIG. 2 is a spacer alignment of LbCAs12a-GSFP crRNA sequences. PAM (protospacer adjacent motif): TTTN, protospace: a spacer sequence.
FIG. 3 shows crRNA detection efficiency of Cas12a fluorescence detection system for GSFP gene specific to bacterial leaf blight of rice. LbCAs12a-GSFP is crRNA designed according to GSFP gene, lbCAs12a-GSFP-BC is ddH 2 O served as a blank. Fluorescence ratio: the ratio of the fluorescence intensity detected by the microplate reader at 5min intervals to the fluorescence intensity detected at 0 min intervals.
FIG. 4 shows the result of agarose gel electrophoresis of RPA primers. M: DL2000;1: RPA-F1R1;2: BC1;3: RPA-F2R2;4: BC2;5: RPA-F3R3;6: BC3; BC: with ddH 2 O is a blank.
Fig. 5 is the result of the optimization of RPA/Cas12a reaction time. A: optimizing RPA reaction time; b: optimization of Cas12a visualization fluorescence detection reaction time; BC: with ddH 2 O is a blank.
FIG. 6 is a sensitivity analysis of the RPA/Cas12a fluorescence detection reaction. A: sensitivity analysis of RPA/Cas12a fluorescence detection reaction. B: sensitivity analysis of PCR detection reaction of GSFP gene. C: sensitivity analysis of the RPA/Cas12a visualized fluorescence detection reaction of GSFP gene. 1-9 in the figure represent respectively: 20 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L, 1 fg/. Mu.L, BC: blank control. Error bars represent standard errors of the signal strength of the three measurements; * P <0.0001; * P <0.001; * P <0.01; * P <0.05.
FIG. 7 is a specific analysis of RPA/Cas12a fluorescence detection reaction. A: specific analysis of RPA/Cas12a fluorescence detection reaction; b: specific analysis of RPA/Cas12a visualized fluorescence detection reactions; BC: with ddH 2 O is a blank.
FIG. 8 is a fluorescent detection reaction field sample detection of RPA/Cas12 a. A: PCR amplification reaction based on GSFP gene of 16 field samples; b:16 field samples are based on the result of visual fluorescence detection of rice bacterial leaf blight of RPA/Cas12 a; n: DNA from healthy leaves was used as negative control; p: DNA of bacterial leaf blight of rice was used as positive control.
FIG. 9 is an RPA/Cas12a-LFA detection reaction. A: preliminary establishment of LFA detection reaction of RPA/Cas12 a; b: specific analysis of the RPA/Cas12a-LFA detection reaction. C: sensitivity analysis of the RPA/Cas12a-LFA detection reaction. 1-9 represent respectively: 20 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L, 1 fg/. Mu.L, BC: blank control. D:16 field samples were tested for bacterial leaf blight of rice based on RPA/Cas12 a-LFA. N: taking DNA of healthy rice leaves as a negative control; p: DNA of bacterial leaf blight of rice was used as positive control.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents used were not manufacturer-identified and were all conventional commercially available products.
The detection operation flow diagram for detecting rice bacterial blight bacteria based on CRISPR/Cas12a provided by the invention is shown in figure 1: firstly, a suspected sample is collected, crude extraction of DNA is carried out, then a target sequence is amplified through an RPA amplification technology, then a CRISPR/Cas12a reaction is carried out, a ssDNA probe is subjected to nonspecific cleavage, and finally a result is read through a fluorescent quantitative PCR instrument, a test paper strip and a blue light gel cutting instrument.
Example 1 target conservation analysis and primer design of specific genes
According to the invention, the rice bacterial blight bacteria specific genes are searched on NCBI websites (https:// www.ncbi.nlm.nih.gov /), and through homology analysis and BLAST on NCBI websites, the rice bacterial blight bacteria specific genes are screened out: glutamine synthetase family protein%GSFP) And (3) a gene.
The crRNA sequence consists of two parts, namely a direct repeated sequence and a spacer sequence. Will beGSFPThe gene sequence is targeted on a CRISPOR (http:// crispor.tefor.net /) website, and in order to ensure the high efficiency of the work of the crRNA sequence, a spacing sequence with high score is selected to design the crRNA. The interval sequence is as follows: ACUGUAGACAUCGCAGAUCAACA. To ensure that most regions of this pathogen can be detected, 19 strains typical of different regions were selected for alignment of spacer sequences by DNAMAN software (FIG. 2). Sending to the biological company to synthesize crRNA sequence: UAAUUUCUACUAAGUGUAGAUACUGUAGACAUCGCAGAUCAACA (SEQ No. 3). Design by Primer 5.0 softwareGSFPRPA and PCR primers for a gene comprising PAM sequences.
PCR primers and RPA primers described in Table 1
Example 2 validation of crRNA sequence and RPA amplification reaction
Validation of 1 crRNA sequence
The target sequence containing PAM sequence was first amplified by PCR reaction. The PCR reaction system is as follows: 12.5 Mu LGreen Master Mix enzyme 1 mu L, GSFP-F/R (10. Mu.M) 1. Mu.L each, 1. Mu.L of genomic DNA template, and ddH 2 O was made up to 25. Mu.L. The PCR reaction program was set as follows: pre-denaturation at 94℃for 3 min; denaturation at 94℃for 30 s, annealing at 58℃for 30 s, extension at 72℃for 45 s for a total of 35 cycles; finally, the mixture is extended for 10 min at 72 ℃ and stored at 4 ℃.
The CRISPR/Cas12a reaction is then performed. 20. Mu L of the reaction system is as follows: 2. mu.L of 10 XBuffer 2.1, 1. Mu.L of crRNA (10. Mu.M), 1. Mu.L of RNase inhibitor (40U/. Mu.L), 2. Mu.L of ssDNA-reporter-FAM (10. Mu.M), 1. Mu.L of LbCAs12a (1. Mu.M), 2. Mu.L of PCR product, 11. Mu.L of DEPC treated water were reacted on a microplate reader (λex: 485 nm; λem: 535 nm), and the fluorescent value was detected every 5 minutes and reacted at 37℃for 1 h. Experiments were repeated three times and plotted using GraphPad Prism 8. Peak was also seen at about 10 min, and peak was seen at about 50 min, with fluorescence ratios of about 3, indicating that crRNA was active (fig. 3). Thus, it can be used in subsequent CRISPR/Cas12a experiments.
2 RPA amplification reaction
The reaction system of RPA 50. Mu.L is: 2.4 mu.L forward primer (10. Mu.M), 2.4. Mu.L reverse primer (10. Mu.M), 29.5. Mu. LRehydration Buffer, 11.2. Mu.L ddH 2 O and 2 mu L of template DNA are mixed uniformly and then are quickly placed into a reaction small tube containing freeze-dried enzyme powder, 2.5 mu L of MgOAC is added on a tube cover after the freeze-dried enzyme powder is dissolved, and the reaction conditions are as follows: the reaction was carried out at 39℃for 20 min. After the RPA reaction is finished, 5 mu L of RPA amplification product and 1 mu L of 6×loading buffer are uniformly mixed and added into a sample adding hole, 2% agarose gel is used, the voltage is set at 120 and V, the agarose gel is taken out after electrophoresis for 30 min, and the agarose gel is photographed and stored in a gel electrophoresis imaging system. The result shows that rice bacterial leaf blight bacteria can be detected only by using the RPA-GSFP-F1/R1 primer for reactionThe target product was 263 bp, which was specifically detected, and the negative control did not produce any bands (fig. 4).
Example 3 optimization of the reaction time for RPA/Cas12a fluorescence detection
Optimization of 1 RPA reaction time
The incubation time was kept constant at 39℃and RPA amplification reactions were performed with the reaction times changed to 5min, 10 min, 15min, and 20 min, respectively (example 2). And adding RPA amplification products reacted at different times into a CRISPR/Cas12a reaction system to perform a cleavage reaction. The CRISPR/Cas12a reaction system is: 2. mu.L SF buffer (Tris-OAC, mgOAC, BSA and DTT), 1. Mu.L crRNA (10. Mu.M), 1. Mu.L RNase inhibitor (40U/. Mu.L), 2. Mu.L ssDNA-reporter-CY5 (10. Mu.M), 1. Mu.L LbCAs12a (1. Mu.M), 2. Mu.L RPA product, 11. Mu.L DEPC treated water, and the reaction was performed on a real-time fluorescent quantitative PCR instrument at 37℃with 1 h. When the RPA reacts for 5min, a CRISPR/Cas12a detection reaction can be caused; when the RPA reaction is about 10 min, the fluorescence signal of the Cas12a reaction can reach a maximum. Thus, the RPA reaction time was set to 10 min in the subsequent experiments (FIG. 5-A).
Optimization of 2 cas12a reaction time
And adding a product obtained after the RPA reaction for 10 min into a CRISPR/Cas12a reaction system, keeping the incubation time unchanged at 37 ℃, and setting the Cas12a cutting reaction time to be 5min, 10 min, 15min and 20 min. The CRISPR/Cas12a reaction system is: 2. mu.L SF buffer (Tris-OAC, mgOAC, BSA and DTT), 1. Mu.L crRNA (10. Mu.M), 1. Mu.L RNase inhibitor (40U/. Mu.L), 2. Mu.L ssDNA-reporter-FAM-2 (10. Mu.M), 1. Mu.L LbCAs12a (1. Mu.M), 2. Mu.L RPA product, 11. Mu.L DEPC treated water. Fluorescence results were observed by a blue light cut gum machine (470 nm). Almost saturated fluorescence can be achieved when the Cas12a cleavage reaction is 5min (fig. 5-B). Therefore, if the Cas12a cleavage reaction result is observed with naked eyes later, the Cas12a reaction time can be set to 5 min.
Finally, combining the optimized CRISPR/Cas12a reaction system with the optimized RPA system to establish an optimal RPA/Cas12a reaction system.
Example 4 establishment of method for rapidly detecting bacterial blight of rice based on CRISPR/Cas12a-RPA
1) Collecting suspected samples of rice bacterial leaf blight bacteria, shearing a leaf midrib region (about 0.5 cm multiplied by 2 cm) of a leaf to be detected by scissors, shearing into fine fragments, placing the fine fragments in a clean 1.5 mL sterile centrifuge tube, adding 500 mu L of 0.5M NaOH solution, mashing the leaf by a sterilization grinding rod, standing for 1 min at room temperature, diluting by 50 times by using TE buffer (Tris-EDTA), and directly taking the diluted fragments as RPA templates.
2) Preparing a solution according to a reaction system: the reaction system of RPA 50. Mu.L is: 2.4 mu.L forward primer (10. Mu.M), 2.4. Mu.L reverse primer (10. Mu.M), 29.5. Mu. LRehydration Buffer, 11.2. Mu.L ddH 2 O and 2 mu L of template DNA are mixed uniformly and then are quickly placed into a reaction small tube containing freeze-dried enzyme powder, after the freeze-dried enzyme powder is dissolved, 2.5 mu L of MgOAC is added on a tube cover, and the tube is quickly centrifuged to the bottom of the tube. The reaction was carried out at 39℃for 10 min.
3) RPA/Cas12a fluorescence detection reaction. 20. Mu L of the reaction system is as follows: 2. mu.L SF buffer (Tris-OAC, mgOAC, BSA and DTT), 1. Mu.L crRNA (10. Mu.M), 1. Mu.L RNase inhibitor (40U/. Mu.L), 2. Mu.L ssDNA-reporter-CY5 (10. Mu.M), 1. Mu.L LbCAs12a (1. Mu.M), 2. Mu.L RPA product, 11. Mu.L DEPC treated water. The reaction was performed on a fluorescent quantitative PCR apparatus with the procedure set to 37℃for 30 s and 37℃for 30 s, and fluorescence was collected every 1 min during the reaction for 60 cycles, which was 1 h in total.
4) RPA/Cas12a visual fluorescence detection reaction: 20. mu L of the reaction system is as follows: 2. mu.L SF buffer (Tris-OAC, mgOAC, BSA and DTT), 1. Mu.L crRNA (10. Mu.M), 1. Mu.L RNase inhibitor (40U/. Mu.L), 2. Mu.L ssDNA-reporter-FAM-2 (10. Mu.M), 1. Mu.L LbCAs12a (1. Mu.M), 2. Mu.L RPA product, 11. Mu.L DEPC treated water. The reaction was carried out at 37℃for 5min, and after the completion of the reaction, the results were observed using a blue light gel cutting instrument.
5) RPA/Cas12a-LFA detection reaction: 20. mu L of the reaction system is as follows: 2. mu.L SF buffer (Tris-OAC, mgOAC, BSA and DTT), 1. Mu.L crRNA (10. Mu.M), 1. Mu.L RNase inhibitor (40U/. Mu.L), 2.5. Mu.L FB-reporter (100 nm), 1. Mu.L LbCAs12a (1. Mu.M), 2. Mu.L RPA product, 10.5. Mu.L DEPC treated water. After the reaction was completed, the mixture was reacted at 37℃for 20 minutes, and after the completion of the reaction, the mixture was made up to 50. Mu.L with DEPC-treated water. The product was dropped onto the test strip binding pad end, and a red band was observed at the quality control line.
Example 5 sensitivity and specificity analysis of detection methods for RPA/Cas12a
1 extraction of DNA: genomic DNA of Rhizoctonia solani was extracted with the Guangzhou Meiger Biotechnology Co.Ltd HiPure Bacterial DNA Kit bacterial genomic DNA extraction kit.
The method comprises the following steps:
1) Taking bacterial liquid 2 mL cultured overnight in a shaking way, transferring the bacterial liquid into a 2 mL sterile centrifuge tube, centrifuging the bacterial liquid at 10000 r/min for 1 min to collect bacteria, and pouring out the residual culture medium as much as possible.
2) 220 mu L Buffer STE Plus, 10 mu L RNaseA and 30 mu L Lysozyme were added to the precipitated bacteria, vortexed, resuspended sufficiently and allowed to stand at room temperature for 10-15 min. To the bacterial suspension was added 10. Mu.L of protease K and 250. Mu.L of Buffer DL, and mixed by vortexing, and water-bath at 70℃for 10 min.
3) 250. Mu.L of absolute ethanol was added to the lysate. Vortex mixing 15 s. If flocculent precipitate appears in the step, the flocculent precipitate is scattered as much as possible by sucking and beating the flocculent precipitate with a liquid-transfering gun for several times.
4) HiPure DNA Mini Columnl is contained in a 2 mL collection tube. Transferring the mixed liquor (including sediment) obtained in the step (4) to a column. 10000 Centrifuging for 1 min at r/min. If the column is blocked, the centrifugation speed is increased to 13000 r/min, and the column is centrifuged for 3 min.
5) The effluent was discarded and the column was packed into a recovery header. 500. Mu.L Buffer GW1 (diluted with ethanol) was added to the column. 10000 Centrifuging for 1 min at r/min.
6) The filtrate was discarded and the column was packed into a recovery header. 650. Mu.L Buffer GW2 (diluted with ethanol into the column 10000 r/min centrifuged for 1 min) was added.
7) The effluent was discarded and the column was packed into a recovery header. 10000 Centrifuging for 2 min at r/min. The column was placed in a new 1.5 mL sterile centrifuge tube. And adding 30-100 mu L of Buffer AE preheated to 70 ℃ to the center of the column membrane, and standing for 3 min.10000 Centrifuging for 1 min at r/min.
8) The DNA binding column was discarded. The extracted standard genome DNA was measured for concentration and quality by an ultra-micro spectrophotometer.
Sensitivity analysis of detection method of 2 RPA/Cas12a
The genomic DNA of rice bacterial leaf blight bacteria was diluted to 20 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L, 1 fg/. Mu.L, and the RPA/Cas12a fluorescence detection reaction and the RPA/Cas12a-LFA detection reaction were performed to achieve sensitivity evaluation, and the test was repeated 3 times (see example 4). PCR experiments were also performed using GSFP-F/R primers, and the sensitivity differences between the two were compared. Preliminary set-up of the RPA/Cas12a-LFA detection reaction is shown in panel A of FIG. 9.
The result shows that the lower limit of detection of rice bacterial blight bacteria by the RPA/Cas12a fluorescence detection reaction is 100 fg/mu L, the sensitivity of the PCR amplification reaction is 1 pg/mu L, and the sensitivity is 10 times higher than that of the PCR reaction; the result is observed by a blue light gel cutting instrument, the lower limit of detection of bacterial blight bacteria of rice by the RPA/Cas12a visual fluorescence detection reaction is 1 fg/mu L, and the sensitivity is 1000 times higher than that of PCR; the lower limit of detection of rice bacterial leaf blight bacteria by RPA/Cas12a-LFA is 100 fg/mu L, which is 10 times higher than the sensitivity of PCR reaction (FIG. 6 and FIG. 9, panel B). The sensitivity of the RPA/Cas12a detection method is significantly better than that of the PCR reaction.
Specific analysis of detection method of 3 RPA/Cas12a
Other strains of the genus xanthomonas, as well as bacterial suspensions of other closely related strains (od=0.8) (see table 2 for strain details) were used as templates for RPA amplification with ddH 2 O is a negative control. The specificity was assessed using the RPA/Cas12a fluorescence detection reaction, and the specificity of the RPA/Cas12a-LFA reaction was similarly assessed.
The results show that when the bacterial strain of the bacterial leaf blight of rice is specifically detected by a fluorescence quantitative PCR instrument, a remarkable fluorescent signal is generated, and the near-edge bacterial strain has weak signal intensity and is relatively different from that of the blank control (A diagram of FIG. 7). When the observation result is visualized by a blue light gel cutting instrument, only rice bacterial blight bacteria can observe remarkable macroscopic fluorescence, and the near-edge strain cannot generate macroscopic fluorescence (B diagram of FIG. 7). When observed through the test strip, only the bacterial strain of rice bacterial leaf blight had no red band at the test line and red band at the quality control line (panel C of fig. 9). Therefore, the fluorescence detection reaction of the RPA/Cas12a has stronger specificity.
TABLE 2 control strains used in the invention
Example 6 field sample testing
16 samples with suspected symptoms are collected from different rice fields around three parts of Hainan province, the collected leaves are subjected to surface disinfection, DNA crude extraction, RPA/Cas12a visual fluorescence detection and test strip analysis, and the test strip analysis is shown in example 4.
First byGFSPThe gene PCR amplification was verified that 11 samples showed positive amplification of rice bacterial leaf blight bacteria and the remaining 5 samples were negative (panel A of FIG. 8). Then carrying out the RPA/Cas12a fluorescence detection visualization detection reaction and the RPA/Cas12a-LFA detection reaction, and carrying out the detection results of 16 samples andGFSPthe results of the gene PCR detection were kept completely identical (panel B in FIG. 8 and panel D in FIG. 9).
It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Claims (8)

1. A reagent combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA is characterized by comprising an RPA primer sequence and a crRNA sequence designed according to GSFP genes,
the RPA primer sequence is as follows:
the forward primer RPA-GSFP-F1 is: 5'-TTGGCTAATGACATGGAAATGGTTCCCGTG-3' the number of the individual pieces of the plastic,
the reverse primer RPA-GSFP-R1 is: 5'-AATTCAAGCTCCGATGCGAAATAGGCACGC-3' the number of the individual pieces of the plastic,
the crRNA sequence is: UAAUUUCUACUAAGUGUAGAUACUGUAGACAUCGCAGAUCAACA;
the detection kit also comprises a ssDNA probe sequence, wherein the ssDNA probe sequence comprises a ssDNA probe sequence for visual fluorescence detection of rice bacterial blight bacteria or a ssDNA probe sequence for detection of a lateral flow chromatography test strip of rice bacterial blight bacteria;
the ssDNA probe sequence ssDNA-reporter-FAM-2 for visual fluorescence detection of rice bacterial leaf blight bacteria is as follows: 5' -6' fam-TGTCTTATcccccATAAGACA-BHQ1-3';
the ssDNA probe sequence FB-reporter for detecting the rice bacterial leaf blight bacteria by using the lateral flow chromatography test strip is as follows: 5' -6' FAM-TTTTTTTTTTT-Biotin-3 '.
2. A detection kit for rapidly detecting bacterial leaf blight of rice, which is characterized by comprising the RPA primer sequence, crRNA sequence, ssDNA probe sequence, rehydration Buffer, mgOAC, lyophilized enzyme powder, lbCAS12a enzyme and ddH as claimed in claim 1 2 O; the freeze-dried enzyme powder is used for RPA reaction; the kit is divided into two reaction systems, including a reaction system for performing an RPA reaction and a reaction system for performing CRISPR/Cas12 a.
3. The kit for rapidly detecting bacterial leaf blight of rice according to claim 2, wherein the kit comprises eight rows of PCR reaction tubes with a plurality of independent units, wherein the PCR reaction tubes contain the lyophilized enzyme powder.
4. The kit for rapid detection of bacterial leaf blight of rice according to claim 3, wherein the reaction system for performing RPA reaction is operated in a total volume of 50 μl: 2.4 mu.L of forward primer RPA-GSFP-F1 at a concentration of 10. Mu.M, 2.4. Mu.L of reverse primer RPA-GSFP-R1 at a concentration of 10. Mu.M, 29.5. Mu. L Rehydration buffer, 11.2. Mu.L of ddH 2 O, 2 mu L template DNA, mixing evenly and then quickQuickly placing the mixture into the PCR reaction tube, dissolving freeze-dried enzyme powder, and finally adding 2.5 mu L of MgOAC; when the reaction system for performing CRISPR/Cas12a is in operation, the reaction system is selected from any one of the following:
the total volume of the reaction system was 20. Mu.L: 2. mu.L SF buffer, 1. Mu.L crRNA sequence at a concentration of 10. Mu.L of enzyme inhibitor with RNase activity of 40U/. Mu.L, 2. Mu.L ssDNA-reporter-FAM-2 at a concentration of 10. Mu.M, 1. Mu.L LbCAs12a enzyme at a concentration of 1. Mu.M, 2. Mu.L RPA reaction product, 11. Mu.L DEPC treated water;
or a total volume of 20. Mu.L: 2. mu.L SF buffer, 1. Mu.L crRNA at a concentration of 10. Mu.M, 1. Mu.L RNase inhibitor with an enzyme activity of 40U/. Mu.L, 2.5. Mu.L FB-reporter at a concentration of 100 nm, 1. Mu.L LbCAs12a enzyme at a concentration of 1. Mu.M, 2. Mu.L RPA reaction product, 10.5. Mu.L DEPC treated water.
5. The use of a detection kit for rapidly detecting bacterial blight of rice as claimed in any one of claims 2 to 4 in detecting bacterial blight of rice in the field.
6. The detection method for rapidly detecting the bacterial leaf blight of the rice is characterized by comprising the following steps of:
1) Extracting genome DNA of a rice bacterial leaf blight suspected sample;
2) Performing an RPA amplification reaction using the kit of claim 2 or 3 using the genomic DNA extracted in step 1) as a template;
3) The kit of claim 2 or 3 is used for performing CRISPR/Cas12a reaction on the RPA amplification product obtained in the step 2) and then performing fluorescence detection or lateral flow chromatography test strip detection.
7. The method for rapidly detecting bacterial blight of rice according to claim 6, wherein,
the method for extracting the genomic DNA of the rice bacterial leaf blight suspected sample in the step 1) comprises the following steps: cutting the leaves of a suspected sample of bacterial leaf blight of rice into fine fragments, placing the fragments in a clean 1.5 mL sterile centrifuge tube, adding 0.5M NaOH solution, mashing the leaves with a sterilized grinding rod, standing for 1 min at room temperature, and diluting by using TE Buffer for 50 times;
in step 2), 50. Mu.L of the reaction system was: 2.4 mu.L of forward primer RPA-GSFP-F1 at a concentration of 10. Mu.M, 2.4. Mu.L of reverse primer RPA-GSFP-R1 at a concentration of 10. Mu.M, 29.5. Mu. L Rehydration Buffer, 11.2. Mu.L of ddH 2 O and 2 mu L of template DNA are mixed uniformly and then are rapidly put into the PCR reaction tube, 2.5 mu L of MgOAC is added on a tube cover after the freeze-dried enzyme powder is dissolved, and the reaction conditions are as follows: amplifying for 10 min at 39 ℃;
in step 3), 20. Mu.L of the reaction system was: 2. mu.L SF buffer, 1. Mu.L crRNA sequence at a concentration of 10. Mu.L RNase inhibitor with an enzyme activity of 40U/. Mu.L, 2. Mu.L ssDNA-reporter-FAM-2 at a concentration of 10. Mu.M, 1. Mu.L LbCAs12a enzyme at a concentration of 1. Mu.M, 2. Mu.L RPA reaction product, 11. Mu.L DEPC treated water under the reaction conditions: reacting for 5min at 37 ℃, and observing a fluorescence result by a 470 nm blue light gel cutting instrument;
or 20. Mu.L of the reaction system: 2. mu.L of SF buffer solution, 1 mu.L of crRNA sequence with the concentration of 10 mu M, 1 mu.L of RNase inhibitor with the enzyme activity of 40U/. Mu.L, 2.5 mu.L of FB-reporter with the concentration of 100 nm, 1 mu.L of LbCAs12a enzyme with the concentration of 1 mu M, 2 mu.L of RPA reaction product, 10.5 mu.L of DEPC treatment water, reacting for 20 min at 37 ℃, supplementing 50 mu.L of DEPC treatment water, uniformly mixing, dripping the mixture into the bonding pad end of a test strip, and observing the colors of a detection line and a quality control line on the test strip.
8. The use of a detection method for rapidly detecting bacterial blight of rice according to claim 6 or 7 in bacterial blight detection of rice.
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