Method for visually and rapidly detecting rhizoctonia cerealis based on ERA-CRISPR / Cas12a
By applying ERA-CRISPR/Cas12a technology in the detection of wheat trefoil blight, the problems of slow detection speed, high cost and insufficient sensitivity in the prior art are solved, and a fast, accurate and low-cost detection effect is achieved.
Patent Information
- Application Number
- CN202510232678.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-05-27
AI Technical Summary
The prior art is difficult to detect wheat graft blight quickly and accurately, especially in field conditions. Traditional methods require professional equipment and high costs, and lack sensitivity and specificity.
The internal transcriptional spacer (ITS) sequence of wheat striatum blight was obtained by RT-ERA amplification, and then the CRISPR/Cas12a fluorescence detection system or test strip detection system was used for detection.
It has achieved rapid detection of wheat striae blight bacteria under constant temperature conditions of 25-40℃. The lower limit of test strip detection is 0.1pg/μL, and the reaction time is only 40 minutes. The detection results can be intuitively reflected, which has the advantages of high sensitivity, strong specificity and short detection time.
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Figure CN120041601A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of agricultural biotechnology. Specifically, the present invention relates to a method for visual and rapid detection of Rhizoctonia cerealis in wheat based on ERA-CRISPR / Cas12a. Background Art
[0002] Wheat sharp eyespot caused by the infection of Rhizoctonia cerealis is an important soil-borne disease in wheat production. In recent years, with the influence of climatic conditions, the change of farming systems such as straw returning to the field, and the improvement of water and fertilizer conditions, wheat sharp eyespot has spread rapidly in the vast wheat-growing areas of China, and the harm has become increasingly serious. It has now become the main disease in the middle and lower reaches of the Yangtze River and the Huanghuai wheat-growing areas, causing serious economic losses every year. Wheat sharp eyespot often occurs mixed with wheat root and stem diseases such as root rot and basal stem rot. Their symptoms are similar in the early stage of disease, which affects the early diagnosis and control of the disease. At present, the control measures for wheat sharp eyespot mainly include soil treatment and chemical seed dressing, supplemented by spraying fungicides such as jinggangmycin, triadimefon, and triadimenol in the early stage of greening. To improve the accuracy and pertinence of chemical control and avoid blindness and waste, establishing a rapid, sensitive and convenient pathogen detection method helps to improve the comprehensive control effect of wheat sharp eyespot.
[0003] At present, regarding the detection methods of Rhizoctonia cerealis in wheat, in addition to traditional morphological identification, there are also molecular biology detection methods. Molecular detection methods include conventional PCR, Real-time PCR, multiplex PCR, and loop-mediated isothermal amplification technology (LAMP). Traditional morphological identification methods are time-consuming and laborious. Conventional PCR and Real-time PCR have the advantages of accurate detection and high sensitivity, but these two detection technologies require high professional qualities of instrument equipment and operators, need special instruments, and have high detection costs, and are not suitable for rapid detection in the field. The LAMP technology can react under the constant temperature condition of 60–65°C, but this technology requires designing 4–6 primers, the primer design is complex, and it is prone to false positives, and cannot meet the rapid detection requirements under field conditions.
[0004] The enzyme-catalyzed recombinase isothermal amplification (ERA) method can amplify specific segments of trace target DNA / RNA by hundreds of millions of times within 7-10 minutes under the constant temperature condition with the optimum temperature of 37°C - 42°C. It does not require complex instruments and is suitable for on-site rapid detection. Cas12a is a type II-V Cas protein. It has no cleavage activity when not activated. When it binds to a specific crRNA, the conformation of Cas12a will change, and it forms a Cas12a-crRNA binary complex with the crRNA. This complex can specifically recognize the target DNA and activate its endonuclease activity. By adding a probe, the presence of the target can be detected by judging whether the probe is cleaved. The combined application of the enzyme-catalyzed recombinase isothermal amplification technology and the Cas detection system can greatly improve the detection sensitivity.
[0005] Wheat sharp eyespot is an important soil-borne disease. Studies have shown that the pathogen of wheat sharp eyespot can survive in the soil for a long time and can be transmitted with seeds. Therefore, the establishment of a rapid detection technology is of great significance for the identification, diagnosis, monitoring and early warning of wheat sharp eyespot. Summary of the Invention
[0006] To solve the problems of rapid, efficient and accurate detection of the pathogen of wheat sharp eyespot, through extensive screening and long-term experiments, the present invention proposes a method for visual rapid detection of the pathogen of wheat sharp eyespot based on ERA-CRISPR / Cas12a, thus completing the present invention. Specifically, the technical solution of the present invention is as follows:
[0007] A method for visual rapid detection of the pathogen of wheat sharp eyespot based on ERA-CRISPR / Cas12a, the method comprising:
[0008] (4) Obtaining the genomic DNA of the pathogen of wheat sharp eyespot;
[0009] (5) Using specific primers to perform RT-ERA amplification on the internal transcribed spacer (ITS) sequence of the pathogen of wheat sharp eyespot to obtain an ERA reaction product;
[0010] (6) Performing visual detection of CRISPR / Cas12a on the ERA reaction product.
[0011] In one embodiment, the RT-ERA reaction is: After fully mixing the solvent, upstream and downstream primers, DNA template, ddH 2 O, adding an initiator, and after mixing evenly, placing the reaction tube at 25°C - 40°C for a period of reaction; preferably, the reaction temperature is 35°C.
[0012] In one embodiment, a CRISPR / Cas12a fluorescence detection system is used to detect the ERA reaction product.
[0013] The CRISPR / Cas12a fluorescence detection system includes: NEB buffer, LbCas12a, RNase inhibitor, crRNA, Nuclease-free water, FQ probe, and ERA reaction product. After mixing the reaction system evenly, it is placed at 37°C for reaction for 30 min.
[0014] Preferably, the CRISPR / Cas12a fluorescence detection system includes: 2 μL of NEB buffer, 1 μL of 1 μmol / L LbCas12a, 1 μL of 40 U / μL RNase inhibitor, 1 μL of 2 μmol / L crRNA, 11 μL of Nuclease-free water, 2 μL of 1 μmol / L FQ probe, and 2 μL of ERA reaction product. After mixing the reaction system evenly, it is placed at 37°C for reaction for 30 min.
[0015] In one embodiment, the ERA reaction product is detected using the CRISPR / Cas12a test strip detection system.
[0016] The CRISPR / Cas12a test strip detection system includes: CRISPR / Cas12a reaction product and Cas12 / 13 nucleic acid test strip; during detection, the CRISPR / Cas12a reaction product is added to the Cas12 / 13 nucleic acid test strip; or the Cas12 / 13 nucleic acid test strip is inserted into a vessel / tube containing the CRISPR / Cas12a reaction product. Among them, the CRISPR / Cas12a reaction product is obtained from the following reaction system: NEB buffer, 1 μmol / L LbCas12a, 40 U / μL RNase inhibitor, 2 μmol / L crRNA, Nuclease-free water, 1 μmol / L FB probe, and ERA reaction product. After mixing the reaction system evenly, it is reacted at 37°C for 10 - 20 min.
[0017] Preferably, the CRISPR / Cas12a reaction product is obtained from the following reaction system: 6 μL of NEB water, 3 μL of 1 μmol / L LbCas12a, 1.5 μL of 40 U / μL RNase inhibitor, 3 μL of 2 μmol / L crRNA, 38.5 μL of Nuclease-free water, 6 μL of 1 μmol / L FB probe, and 2 μL of ERA reaction product. After mixing the reaction system evenly, it is reacted at 37°C for 10 - 20 min; more preferably, the reaction time at 37°C is 15 min.
[0018] In a second aspect of the present invention, the present invention provides a kit for visual and rapid detection of *Rhizoctonia cerealis* based on ERA-CRISPR / Cas12a, which includes: (1) genomic DNA extraction reagent for *Rhizoctonia cerealis*; (2) RT-ERA amplification reagent for the internal transcribed spacer (ITS) sequence of *Rhizoctonia cerealis*; (3) visual detection reagent for ERA-CRISPR / Cas12a of the RT-ERA amplification product.
[0019] In one embodiment, the RT-ERA amplification reagent includes primers specific to the internal transcribed spacer (ITS) sequence of *Rhizoctonia cerealis*; the primer sequences are as shown in SEQ ID NO.X and SEQ ID NO.X.
[0020] In one embodiment, the visual detection reagent for ERA-CRISPR / Cas12a is a fluorescence detection reagent; the fluorescence detection reagent includes: NEB buffer, LbCas12a, RNase inhibitor, crRNA, Nuclease-free water, FQ probe; the crRNA sequences are as shown in SEQ ID NO.X and SEQ ID NO.X, and the FQ probe is as shown in SEQ ID NO.X.
[0021] In one embodiment, the visual detection reagent for ERA-CRISPR / Cas12a is a test strip detection reagent; the test strip detection reagent includes: CRISPR / Cas12a reaction reagent and Cas12 / 13 nucleic acid test strip; the CRISPR / Cas12a reaction reagent includes: NEB buffer, LbCas12a, RNase inhibitor, crRNA, Nuclease-free water, FB probe; the crRNA sequences are as shown in SEQ ID NO.X and SEQ ID NO.X, and the sequence of the FB probe is as shown in SEQ ID NO.X.
[0022] In a third aspect of the present invention, the present invention provides the application of the kit for visual and rapid detection of *Rhizoctonia cerealis* based on ERA-CRISPR / Cas12a in the detection of *Rhizoctonia cerealis*.
[0023] In one embodiment, the kit is a CRISPR / Cas12a fluorescence detection kit.
[0024] In one embodiment, the kit is a CRISPR / Cas12a test strip detection kit. In the test strip detection, under natural light, if both the quality control line and the detection line show visible red bands to the naked eye, it is determined as a positive result; if the quality control line shows a red band and the detection line does not develop color, it is determined as a negative result.
[0025] Beneficial effects
[0026] (1) The present invention has established a visual rapid detection system and kit for Rhizoctonia cerealis in wheat based on the principle of ERA-CRISPR / Cas12a, laying a technical foundation for the rapid diagnosis, infection prevention and control of wheat sharp eyespot.
[0027] The technical method of the present invention can be carried out under the constant temperature condition of 25-40°C. The detection limit of the test strip is 0.1 pg / μL (the sensitivity of the fluorescence detection system can reach 0.01 pg / μL). The reaction time only needs 40 minutes. The detection result can be directly reflected by means of a nucleic acid test strip. It has the advantages of strong specificity, high sensitivity, short detection time, visualization, etc., and is not limited by materials, sites and instruments. It can detect the target pathogen from complex samples such as plants, seeds and soils, providing guidance for the large-scale popularization and application of this technology. Brief description of the drawings
[0028] Figure 1 It is the screening result of ERA primers. Among them, M: DNA Maker; 1: Rhizoctonia cerealis; 2: Bipolaris sorokiniana; 3: Fusarium pseudograminearum; 4: Fusarium graminearum; 5: Fusarium verticillioides; 6: Fusarium oxysporum; 7: negative control NC.
[0029] Figure 2 It is the optimization result of ERA reaction temperature. Among them, M: DNA Maker; 1: 25°C; 2: 30°C; 3: 35°C; 4: 40°C.
[0030] Figure 3 It is the screening of crRNA primers and crRNA concentration. Among them, A. Screening of crRNA primers. B. Screening of crRNA concentration in the fluorescence system. C. Screening of crRNA concentration in the test strip system, N: negative; P: positive; C: control line; T: test line.
[0031] Figure 4 It is the optimization of ssDNA-FB concentration by the test strip method. Among them, A. Screening of ssDNA-FB concentration range; B. Screening of the optimal concentration of ssDNA-FB, N: negative; P: positive; C: control line; T: test line.
[0032] Figure 5For the optimization of the cleavage time of ERA-CRISPR / Cas12. Among them, A. Optimization of the cleavage time of the ERA-CRISPR / Cas12 fluorescence system; B. Optimization of the cleavage time of the ERA-CRISPR / Cas12 test strip method.
[0033] Figure 6 For the specificity verification of the ERA-CRISPR / Cas12a detection system. Among them, A: Fluorescence detection results; B: Test strip detection results, N: Negative; P: Positive; C: Control line; T: Test line.
[0034] Figure 7 For the sensitivity evaluation (pg / μL) of the ERA-CRISPR / Cas12a detection system. Among them, A: Fluorescence detection results; B: Test strip detection results, N: Negative; P: Positive, C: Control line, T: Test line.
[0035] Figure 8 For the application of the ERA-CRISPR / Cas12a detection system in actual samples. Among them, A: Detection of hyphae in soil; B: Detection of hyphae in seeds; N: Negative; P: Positive; C: Control line; T: Test line. Detailed implementation mode
[0036] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0037] Example 1:
[0038] 1. Materials and methods
[0039] 1.1 Materials
[0040] The tested strains Rhizoctonia cerealis, Bipolaris sorokiniana, Fusarium graminearum, Fusarium pseudograminearum, Fusarium Verticillioides and Fusarium oxysporum are all preserved in this laboratory, and the above strains have completed morphological and molecular biological identifications.
[0041] 1.2 Methods
[0042] 1.2.1 Genomic DNA Extraction
[0043] The test strains were inoculated on PDA medium and cultured at 25 °C for 4 days to collect mycelia. Genomic DNA was extracted by the CTAB method. And the concentration of DNA was measured.
[0044] 1.2.2 Target Gene Selection and Primer Design
[0045] Using the internal transcribed spacer ITS (GenBank database accession number JQ768030.1) of Rhizoctonia cerealis as the target gene, 3 pairs of ERA primers were designed using Primer primer5. The primer sequences are shown in Table 1. Three pairs of crRNA primers were designed using the online website for crRNA primers (http: / / www.rgenome.net / cas-designer / ), and the T7 promoter sequence was added to their 3' ends. The HiScribe T7 High Yield RNA Synthesis Kit was used to prepare the crRNA template by in vitro transcription. Fluorescent probe FQ and test strip probe FB. The primers and probes were synthesized by General Biosciences Co., Ltd.
[0046] Table 1: ERA Primer, crRNA and Probe Sequences
[0047]
[0048]
[0049] 1.2.3 ERA Amplification System
[0050] According to the instructions of the RT-ERA Basic Amplification Kit (product number KS101) from Suzhou Xianda Gene, 2 pairs of primers were screened. The amplification system was 50 μL: 20 μL of dissolving agent, 2.5 μL each of upstream and downstream primers (10 μmol / L), 3 μL of DNA template, ddH 2 O20 μL. After mixing well, it was transferred to the freeze-dried amplification enzyme. After the dry powder was dissolved, 2 μL of initiator was added. After mixing evenly, the reaction tube was placed at 37 °C for 20 min.
[0051] 1.2.4 Establishment of CRISPR / Cas12a Detection System
[0052] CRISPR / Cas12a fluorescence detection system: 2 μL of NEB buffer, 1 μL of 1 μmol / L LbCas12a, 1 μL of 40 U / μL RNase inhibitor, 1 μL of 2 μmol / L crRNA, 11 μL of Nuclease-free water, 2 μL of 1 μmol / L ProbeFQ, 2 μL of ERA reaction product. After mixing the reaction system evenly, place it at 37 °C for reaction for 30 min.
[0053] CRISPR / Cas12a test strip detection system: NEB buffer 2.1 6 μL, 1 μL of 1 μmol / L LbCas12a, 1.5 μL of 40 U / μL RNase inhibitor, 3 μL of 2 μmol / L crRNA, 38.5 μL of Nuclease-free Water, 6 μL of 1 μmol / L Probe FB, 2 μL of ERA reaction product. After mixing the reaction system evenly, react at 37 °C for 10 - 20 min. Insert the binding pad end of the Cas12 / 13 special nucleic acid test strip (Xianda Gene Biotechnology Co., Ltd.) into the reaction tube and interpret the results. Under natural light, if both the quality control line and the test line show visible red bands, it is judged as a positive result; if the quality control line shows a red band and the test line does not show color, it is judged as a negative result.
[0054] 1.2.5 Optimization of the ERA-CRISPR / Cas12a detection system
[0055] ERA primer screening: Add the 2 designed upstream and downstream ERA primers to the ERA amplification system respectively. Immediately add 6×Loading Buffer and mix the samples evenly. Incubate at 56 °C for 5 min. Then take 10 μL and run it on a 1.5% agarose gel for verification. Screen out the clearest band and select this primer for subsequent experiments.
[0056] Optimization of ERA reaction time and reaction temperature: According to the above system and the selected optimal primer, set the water bath temperature to 25 °C, 30 °C, 35 °C, 40 °C respectively for reaction for 20 min and set ddH2O as the negative control. After the reaction is completed, perform electrophoresis with 1.5% agarose gel electrophoresis. Select the combination with the brightest and clearest band as the optimal reaction time and reaction temperature.
[0057] crRNA screening: Add the 3 designed crRNAs to the CRISPR / Cas12a system respectively. Compare the cleavage efficiency of different crRNAs in fluorescence detection, screen out the optimal crRNA sequence from them, and set ddH2O as the negative control.
[0058] Optimization of crRNA concentration: The crRNA concentrations were set at 10 μmol / L, 2 μmol / L, 1 μmol / L, 500 nmol / L, 250 nmol / L, 100 nmol / L, and 50 nmol / L for the experiments of the CRISPR / Cas12a fluorescence and test strip detection systems. The fluorescence status and the color development of the test strip detection line were observed under a blue light gel imager, and the experiments were set with 3 replicates.
[0059] Optimization of CRISPR / Cas12a cleavage time: The CRISPR cleavage times were set at 0, 10, 20, 30 min, and 40 min for fluorescence detection. They were set at 0, 10, 15, and 20 min for test strip detection. According to the strength of the fluorescence signal and whether the test strip detection line showed color at different reaction times, the optimal cleavage time was screened out, and the experiments were set with 3 replicates.
[0060] 1.2.6 Specificity verification of the ERA-CRISPR / Cas12a detection system
[0061] All the DNA of the tested strains that had been extracted was used as a template, and the optimized ERA-CRISPR / Cas12a system was used for detection, with ddH2O as a control, and the experiments were set with 3 replicates. According to the strength of the fluorescence signal and whether the test strip detection line showed color, it was judged whether the system was specific, and the experiments were set with 3 replicates.
[0062] 1.2.7 Sensitivity evaluation of the ERA-CRISPR / Cas12a detection system
[0063] Genomic DNA of Rhizoctonia cerealis at concentrations of 1000, 100, 10, 1, 10 -1 、10 -2 、10 -3 、10 -4 pg / μL were respectively taken as templates, and the optimized ERA-CRISPR / Cas12a system was used for detection, with ddH2O as a control, and the experiments were set with 3 replicates. According to the strength of the fluorescence signal and whether the test strip detection line showed color, the lower detection limit of the system was judged.
[0064] 1.2.8 Application of the ERA-CRISPR / Cas12a system
[0065] To verify the detection effect of the ERA-CRISPR / Cas12a system on bacteria-infected soil, 0.05 g of Rhizoctonia cerealis mycelium was added to 0.25 g of soil. After releasing the DNA in the soil, it was verified using the ERA-CRISPR / Cas12a detection system, with sterile soil as the blank control, and the experiment was set up with 3 replicates. To verify the detection effect of the ERA-CRISPR / Cas12a system on bacteria-infected seeds, a suspension of Rhizoctonia cerealis mycelium was prepared, dropped on sterile wheat seeds, dried at room temperature for 2 days, and verified using the ERA-CRISPR / Cas12a detection system, with sterile seeds as the blank control, and the experiment was set up with 3 replicates.
[0066] 2 Results
[0067] 2.1 Optimization of the ERA amplification system
[0068] 2.1.1 Screening of ERA primers
[0069] Two pairs of ERA primers were screened through the ERA amplification system. After the reaction ended, the products were purified, and then primers were screened using agarose gel electrophoresis. It can be seen from the electrophoresis pattern that the primer pair ERA-F1 and ERA-R1 can amplify a 256-bp band consistent with the size of the target fragment and is specific to Rhizoctonia cerealis (see Figure 1 ). The primers ERA-F1 and ERA-R1 were selected as the optimal primers for subsequent experiments.
[0070] 2.1.2 Optimization of the ERA system reaction temperature
[0071] The ERA reaction system was placed in a water bath at temperatures of 25 °C, 30 °C, 35 °C, and 40 °C for 20 min and then subjected to gel electrophoresis. As Figure 2 shown, the reaction can occur under the conditions of 25 °C - 40 °C. However, the band is the brightest at 35 °C. Therefore, 35 °C was selected as the optimal reaction temperature for ERA for subsequent experiments.
[0072] 2.2 Optimization of the ERA-CRISPR / Cas12a detection system
[0073] 2.2.1 Screening of crRNA primers and the optimal concentration of crRNA
[0074] The crRNA primers were screened through the ERA-CRISPR / Cas12a fluorescence detection system, with water as the negative control, and the results were observed under a blue light gel imager. The results showed that the fluorescence signal of crRNA2 was significantly higher than that of crRNA1 and crRNA3 ( Figure 3 A in), indicating that the cleavage efficiency of crRNA2 in this reaction system was relatively high. Therefore, crRNA2 was selected for subsequent experiments.
[0075] The crRNA concentrations were set at 10 μmol / L, 2 μmol / L, 1 μmol / L, 500 nmol / L, 250 nmol / L, 100 nmol / L, and 50 nmol / L and added to the fluorescence detection system and the test strip detection system respectively to screen the crRNA primers. The results showed that fluorescence could be detected when the crRNA concentration was 500 nmol / L, and the obvious fluorescence intensity reached the strongest at 1 μmol / L( Figure 3 as shown in B). However, in the test strip system, there was only a faint band for 1 μmol / L of crRNA, and the band was most obvious when the crRNA concentration reached 2 μmol / L( Figure 3 as shown in C). In summary, the optimal crRNA concentration for ERA-CRISPR / Cas12a was 2 μmol / L.
[0076] 2.2.2 Optimization of ssDNA concentration by test strip method
[0077] Only when the ssDNA reporter molecule in the test strip is within a suitable concentration range will the test line not appear or be extremely light in color. Therefore, when screening for the optimal ssDNA-FB concentration, to avoid false positives and increase the credibility of the detection results, in this experiment, ssDNA-FB was diluted in concentration, and other components in the CRISPR system were replaced with ddH2O. The ssDNA-FB concentration was set at 10 μmol / L, 5 μmol / L, 1 μmol / L, 500 nmol / L, 250 nmol / L, 100 nmol / L, and 50 nmol / L to observe the optimal concentration at which no band appears in the test line. Through experiments, it was proved that the test line disappeared at a probe concentration of 1 μmol / L. To screen for the optimal ssDNA-FB concentration, probe concentrations of 1 μmol / L, 500 nmol / L, 100 nmol / L, and 50 nmol / L were set for screening, and ddH2O was used to replace ssDNA-FB as a negative control. The test strip detection results showed that the test strip band was clear when the lowest concentration of ssDNA-FB was 100 nmol / L. Therefore, 100 nmol / L was the optimal concentration of ssDNA-FB. 2.2.3 Optimization of the cleavage time of ERA-CRISPR / Cas12a
[0078] The time of the fluorescence method was optimized. The incubation time was set at 0 min, 10 min, 20 min, 30 min, and 40 min. After the reaction ended, the results were observed under a blue light gel imager. As Figure 5 shown in A, fluorescence could already be detected after incubating for 10 min, and the fluorescence intensity was the brightest at 30 min. Therefore, the cleavage time of the CRISPR / Cas12a fluorescence method was optimized to 30 min.
[0079] Using the established test strip system, the time of the test strip was optimized, such as Figure 5 As shown in B of : There was a weak band at 10 min of incubation, and the band was obvious at 15 min of incubation, and the color of the band was the same as that at 25 min and 30 min. Therefore, the cutting time of the CRISPR / Cas12a test strip was optimized to 15 min.
[0080] 2.3 Specificity verification of the ERA-CRISPR / Cas12a detection system
[0081] Using the DNA of all the tested strains as templates for ERA-CRISPR / Cas12a detection, the results showed that only Rhizoctonia cerealis could produce obvious fluorescence signals, and no fluorescence signals could be detected in the rest of the groups. The results of the CRISPR / Cas12a test strip showed that only the test strip of Rhizoctonia cerealis was positive, and the rest were negative results (see Figure 6 ). It indicates that the system established in this study can specifically detect Rhizoctonia cerealis.
[0082] 2.4 Sensitivity evaluation of the ERA-CRISPR / Cas12a detection system
[0083] Using the ERA primer to amplify the DNA of Rhizoctonia cerealis at different dilution gradients and performing CRISPR / Cas12a detection, the results showed that obvious fluorescence signals could be produced at 10 -2 pg / μL and above, while no fluorescence signal could be detected at 10 -3 pg / μL (see A in Figure 7 ), indicating that the detection limit of the fluorescence detection system was 10 -2 pg / μL. The lowest DNA concentration that the test strip could detect was 10 -1 pg / μL. No band was observed in the test strip detection line of the 10 -2 pg / μL test group, which was a negative result (see B in Figure 7 ), indicating that the detection limit of the test strip detection system was 0.1 pg / μL and it had high sensitivity.
[0084] 2.5 Application of the ERA-CRISPR / Cas12a system
[0085] The results showed that the ERA-CRISPR / Cas12a system could detect the samples of Rhizoctonia cerealis hyphae in soil samples, indicating that this detection system could be used to detect pathogenic bacteria in complex soil samples. The ERA-CRISPR / Cas12a detection system could also detect seed-carrying bacteria (seeFigure 8 )。
[0086] In summary, the present invention uses upstream and downstream primers of ERA to perform ERA amplification reaction on target genes to obtain ERA amplification products; an ERA-Cas12a detection system is established using ERA amplification products, Cas12a protein, crRNA and nucleic acid probes, and an ERA-Cas12a reaction product is obtained after the reaction, and is identified by fluorescence detection method or test strip detection method. The detection method developed by the present invention has the advantages of simplicity, rapidity, strong practicability, high sensitivity and strong specificity, and can quickly distinguish from other stem base diseases such as root rot and stem base rot at the early stage of wheat stem disease, and is a very promising molecular diagnosis method.
[0087] The above content is a further detailed description of the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should all be regarded as belonging to the protection scope determined by the claims submitted by the present invention.
Claims
1. A method for rapid visualization detection of wheat sheath blight pathogen based on ERA-CRISPR / Cas12a, characterized in that: The method comprises: Obtain the genomic DNA of wheat sheath blight pathogen; The internal transcribed spacer (ITS) sequence of wheat sheath blight pathogen was amplified by RT-ERA using specific primers to obtain ERA reaction products. Visual detection of CRISPR / Cas12a in ERA reaction products.
2. The method according to claim 1, characterized in that The RT-ERA amplification is as follows: the solvent, upstream and downstream primers, DNA template, and ddH2O are fully mixed, and then the initiator is added. After mixing evenly, the reaction tube is placed at 25°C-40°C for a period of time; preferably, the reaction temperature is 35°C.
3. The method according to claim 1, characterized in that The ERA reaction product is detected using a CRISPR / Cas12a fluorescence detection system; the CRISPR / Cas12a fluorescence detection system includes: NEB buffer, LbCas12a, RNase inhibitor, crRNA, Nuclease-free water, FQ probe, and ERA reaction product.
4. The method according to claim 1, characterized in that: The ERA reaction products were detected using the CRISPR / Cas12a test strip detection system. In the test strip test, if both the quality control line and the test line showed red strips visible to the naked eye under natural light, it was determined to be a positive result; if a red strip appeared on the quality control line and the test line did not show color, it was determined to be a negative result.
5. The method according to claim 4, characterized in that The CRISPR / Cas12a test strip detection system includes: CRISPR / Cas12a reaction products and Cas12 / 13 nucleic acid test strips; during detection, the CRISPR / Cas12a reaction products are added to the Cas12 / 13 nucleic acid test strips; or the Cas12 / 13 nucleic acid test strips are inserted into a vessel / test tube containing the CRISPR / Cas12a reaction products.
6. The method according to claim 5, characterized in that The CRISPR / Cas12a reaction product is obtained from the following reaction system: NEB buffer, 1 μmol / L LbCas12a, 40 U / μL RNase inhibitor, 2 μmol / L crRNA, Nuclease-free water, 1 μmol / L FB probe, ERA reaction product. The reaction system is mixed evenly and reacted at 37°C for 10-20 min; more preferably, the reaction time at 37°C is 15 min.
7. A kit for rapid visualization detection of wheat sheath blight pathogen based on ERA-CRISPR / Cas12a, characterized in that: The kit comprises: (1) a genomic DNA extraction reagent of wheat sheath blight pathogen; (2) an RT-ERA amplification reagent of an internal transcribed spacer (ITS) sequence of wheat sheath blight pathogen; and (3) an ERA-CRISPR / Cas12a visualization detection reagent for the RT-ERA amplification product.
8. The kit according to claim 7, characterized in that The RT-ERA amplification reagent includes primers specific to the internal transcribed spacer (ITS) sequence of wheat sheath blight pathogen; the primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.
2.
9. The kit according to claim 7, characterized in that The visualization detection reagent of ERA-CRISPR / Cas12a is a fluorescent detection reagent; the fluorescent detection reagent includes: NEB buffer, LbCas12a, RNase inhibitor, crRNA, Nuclease-free water, FQ probe; the crRNA sequence is shown in SEQ ID NO.7 and SEQ ID NO.8, and the FQ probe is shown in SEQ ID NO.
12.
10. The kit according to claim 7, characterized in that The visual detection reagent of ERA-CRISPR / Cas12a is a test strip detection reagent; The test strip detection reagent includes: CRISPR / Cas12a reaction reagent and Cas12 / 13 nucleic acid test strip; CRISPR / Cas12a reaction reagent includes: NEB buffer, LbCas12a, RNase inhibitor, crRNA, Nuclease-free water, FB probe; the crRNA sequence is shown in SEQ ID NO.7 and SEQ ID NO.8, and the sequence of the FB probe is shown in SEQ ID NO.11.