Kit for rapidly detecting pathogenic bacteria of kiwifruit canker

Abstract

The invention discloses a rapid detection kit for pathogenic bacteria of kiwifruit canker, which comprises a HopZ5 specific primer, an LbCas12a protein, HopZ5 crRNA, an FQ reporter molecule probe or an FB reporter molecule and a buffer solution, and after the LbCas12a protein specifically cuts a HopZ5 amplification product, detection is carried out through a fluorescence or lateral flow test strip. By combining with an isothermal amplification technology, the sensitivity of the detection method is greatly improved, and the detection method can be used in fields and quarantine sites, has the advantages of high sensitivity, good specificity, rapidness, low equipment requirements and the like, and is conveniently used for field disease investigation and accurate and rapid detection, identification and diagnosis of pathogenic bacteria of kiwifruit canker in primary laboratories.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture and plant quarantine, and particularly relates to a rapid detection kit for pathogenic bacteria of kiwifruit canker. Background Art

[0002] Kiwifruit canker is the most important disease in kiwifruit production and is known as the cancer of kiwifruit. Once the orchard is infected, it cannot be recovered and will cause the orchard to be destroyed and the production to be cut off, which will cause a devastating blow to the entire kiwifruit industry. Kiwifruit canker is caused by Pseudomonas syringae pv. actinidiae (Psa). At present, there is no effective means to prevent and treat kiwifruit canker, so the rapid and accurate identification of pathogens is the most effective and limited way to prevent and treat canker. Conventional methods for identifying pathogens of kiwifruit canker include culture counting combined with PCR and first-generation sequencing, which are time-consuming, labor-intensive, and costly. Recently, a detection method based on antibody-antigen reaction of HopZ5 has also been developed, but its accuracy is low. Therefore, the development of an efficient, accurate, and convenient detection method is very critical for the rapid, accurate, and timely identification of kiwifruit canker, and is very necessary for the early prevention and treatment of kiwifruit canker.

[0003] Cas proteins derived from the microbial immune system are a large class of nucleases, among which Cas12a of subtype II-V has extremely high activity of target-activated trans-single-stranded DNA cleavage activity in addition to RNA-guided specific double-stranded DNA cleavage activity. Multiple studies have established a fast, efficient and convenient nucleic acid detection platform using the trans-single-stranded DNA cleavage activity of Cas12a. This also provides an effective solution for the rapid identification of kiwifruit canker pathogens. However, so far, there is no rapid identification method for kiwifruit canker based on Cas12a, so it is very necessary to establish a rapid detection method based on Cas12a. Summary of the invention

[0004] Aiming at the problem that the pathogenic bacteria of kiwifruit canker disease is difficult to detect quickly, efficiently and accurately, the present invention provides a kit for detecting the pathogenic bacteria of kiwifruit canker disease with high sensitivity, strong specificity, rapidity and visualization.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A rapid detection kit for kiwifruit canker pathogenic bacteria, comprising a HopZ5 specific primer, an LbCas12a protein, a HopZ5 crRNA, an FQ reporter molecule probe or an FB reporter molecule, and a buffer;

[0007] The sequence of the HopZ5 crRNA is shown in SEQ ID NO: 1-6, including any one or more thereof;

[0008] The sequences of the HopZ5 specific primers are shown in SEQ ID NOs: 7 and 8;

[0009] The components of the buffer solution include HEPES, KCl and MgCl2, and the pH value is 6.8-8.8. Preferably, the buffer solution includes 40 mM HEPES (6.8), 60 mM KCl, 6 mM MgCl2, and the pH value is 6.8.

[0010] The method for detecting kiwifruit canker pathogens by the above kit: using HopZ5 specific primers to obtain HopZ5 amplification products through recombinase polymerase amplification, LbCas12a protein specifically cuts the HopZ5 amplification products, and then identifies Pseudomonas syringae pv. actinidiae (Psa) by fluorescence (such as microplate reader, fluorescence meter, blue light gel cutter, fluorescent flashlight) or lateral flow test strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a comparison of the fluorescence detection results of different Psa target sequences. The horizontal axis in the figure represents different guide RNAs, which are HopZ5 crRNA1, crRNA2, crRNA3, crRNA4, crRNA5, and crRNA6, respectively. No crRNA means no guide RNA, which is a negative control.

[0012] Figure 2 These are the optimization results of different concentrations of LbCas12a. The concentration of LbCas12a was set to 50, 100, and 150 nM, where NoCas12a means that no LbCas12a protein was added, which is a negative control.

[0013] Figure 3 It is the optimization result of different reaction buffers.

[0014] Figure 4 This is the optimization result of different crRNA concentrations. The crRNA concentration was set to 20, 40, and 60 nM, where No crRNA means no guide RNA, which is a negative control.

[0015] Figure 5The results of the amplification of HopZ5 at different concentrations are shown in Figure 1. The abscissa in the figure is the amplification of HopZ5 at different concentrations, which are 0.4, 0.04, 0.004, 0.0004, 0.00004, and 0.000004 pM, respectively. No target means that no amplification product is added, which is a negative control.

[0016] Figure 6 This is the result of the blue light gel cutting instrument test. The PCR tube on the left is the experimental group, and the PCR tube on the right is the control group.

[0017] Figure 7 This is the result of the portable fluorescent flashlight test. The PCR tube on the right is the experimental group, and the PCR tube on the right is the control group.

[0018] Figure 8 It is the result of lateral flow test strip test.

[0019] Fig. 9 It is the test result of natural samples. DETAILED DESCRIPTION

[0020] Sources of biological materials used in the present invention:

[0021] 1. LbCas12a protein was expressed and purified by prokaryotic expression;

[0022] 2. All primers, fluorescent reporter molecules, and FB reporter molecules were designed by the inventors and commissioned to be synthesized by Shanghai Bio-Tech Co., Ltd.;

[0023] 3. All PCR polymerases used were purchased from Beijing Quanshijin Co., Ltd.;

[0024] 4. HopZ5 crRNA was prepared using the T7 in vitro transcription kit from NEB;

[0025] 5. Other chemical reagents were purchased from Beijing Dingguo Biotechnology Company;

[0026] 6. Lateral flow test strips were purchased from Suzhou Xianda Gene Biology Company;

[0027] 7. Use TwistAmp Basic kit purchased from NEB.

[0028] The technical solution of the present invention is further described below in conjunction with embodiments, but it should not be construed as limiting the present invention:

[0029] Example 1 Construction and optimization of a rapid detection kit for kiwifruit canker pathogens and its use method

[0030] This example is used to illustrate the construction and optimization process of a rapid detection kit for kiwifruit canker pathogens and a method for using the kit, and to demonstrate that the present invention is used for rapid, efficient and accurate detection of kiwifruit canker pathogens.

[0031] A. DNA extraction of samples to be tested: The total DNA of kiwifruit leaves with ulcer disease symptoms was extracted using the modified CTAB method;

[0032] B. Nucleic acid amplification: HopZ5-specific primers were used and recombinase polymerase (RPA) isothermal amplification system was added to obtain HopZ5-specific amplification products. The sequences of HopZ5-specific primers are as follows: HopZ5-F: 5′-CCGCAATCAGGATGACGTTCGCACAATGTTAATC-3′ (see SEQ ID NO: 7), HopZ5-R: 5′-CGAGCTCTGGCTGTTCTTCAATGGCATCGTTTATTG-3′ (see SEQ ID NO: 8); isothermal amplification reaction was carried out according to the instructions of TwistAmp Basic kit: 29.5 μL rehydration solution, 8.65 μL RNase free H2O, 2.4 μL forward primer and 2.4 μL reverse primer were mixed and added to the dry powder, 2.5 μL magnesium acetate was added after dissolution, 10 μL of the reaction solution was taken into 4 0.2 mL PCR tubes, 1 μL of the extracted total DNA was added, and the reaction was carried out at 37°C for 30 min to obtain the amplification product;

[0033] C. Comparison of detection effects of different HopZ5 crRNAs: The detection reaction solution was prepared according to the following system: 10×Buffer (20mM HEPES (pH 7.4), 60mM NaCl, 6mM MgCl2) 2μL, LbCas12a (1μM) 1μL, HopZ5 crRNA (10μM) 1μL, FQ reporter (FAM-TTATT-BHQ1, 10μM) 1μL, HopZ5 amplification product 1μL (amplification product obtained in step B), H2O (RNAase free) was supplemented to 20μL; 6 kinds of crRNAs were designed, and their sequences were shown in SEQ ID NO: 1-6. An equal amount of RNase free H2O was added to the negative control; after the components of the rapid detection kit for the pathogenic bacteria of kiwifruit canker were added, the reaction was carried out at 37° C. for 30 min;

[0034] D. Fluorescence intensity detection: The reaction solution obtained in the previous step was subjected to fluorescence intensity detection in an ELISA instrument, wherein the excitation light wavelength was 485 nm and the emission light wavelength was 535 nm;

[0035] E. Result analysis: Statistical analysis showed that the fluorescence intensity of the experimental group with different HopZ5 crRNAs added was significantly higher than that of the negative control. Therefore, the six crRNAs used in this kit are highly specific ( Figure 1 );

[0036] F. Optimization of LbCas12a concentration: The reaction solution was prepared according to step C, and crRNA2 was selected, but the amount of LbCas12a used was changed (50nM, 100nM, 150nM). It was found that LbCas12a still had extremely high activity at 50nM ( Figure 2 );

[0037] G. Optimization of reaction buffer of the kit: Prepare the reaction solution according to step C, select crRNA2, but change the reaction buffer (see Table 1), and explore the effect of different buffers on the performance of this kit; the results show that Buffer 7 has the highest reaction activity ( Figure 3 );

[0038] Table 1 Composition of different buffers

[0039]

[0040] H. Optimization of the concentration of HopZ5 crRNA: The reaction solution was prepared according to step C, and crRNA2 was selected. However, the amount of HopZ5 crRNA used was changed (20nM, 40nM, 60nM). The results showed that crRNA still had extremely high activity at 20nM ( Figure 4 );

[0041] I. Optimization of target nucleic acid concentration: The reaction solution was prepared according to step C, and crRNA2 was selected. However, the amount of Hop5 amplification product used was changed (0.4pM, 0.04pM, 0.004pM, 0.0004pM, 0.00004pM, 0.000004pM). The results showed that the detection resolution was extremely high at 0.04pM ( Figure 5 ).

[0042] Example 2 Blue light cutting instrument detection method

[0043] This example is used to illustrate the visualization detection method of a rapid detection kit for kiwifruit canker pathogens using a blue light gel cutting instrument.

[0044] a. First, a reaction system was prepared in a 0.2 mL PCR tube according to step C in Example 1, wherein HopZ5 crRNA is one or more of crRNA1-6; after the preparation was completed, the reaction was carried out at 37° C. for 30 min.

[0045] b. Place the PCR tube in a on a blue light gel cutting instrument for observation; Figure 6 As shown, the PCR tube on the left is the experimental group, and the PCR tube on the right is the control group. Obvious fluorescence can be observed in the experimental group under the naked eye.

[0046] Example 3 Portable fluorescent flashlight detection method

[0047] This example is used to illustrate the use of a portable fluorescent flashlight visualization detection method in a rapid detection kit for kiwifruit canker pathogens.

[0048] i. First, the reaction system of the kit was prepared in a 0.2 mL PCR tube according to step C in Example 1, wherein HopZ5 crRNA is one or more of crRNA1-6; after the preparation was completed, the reaction was carried out at 37° C. for 30 min.

[0049] ii. Use a portable fluorescent flashlight to illuminate the above PCR tube and observe; Figure 7 As shown, the PCR tube on the left is the experimental group, and the PCR tube on the right is the control group. Obvious fluorescence can be observed in the experimental group under the naked eye.

[0050] Example 4 Lateral flow test strip detection method

[0051] (1) The detection reaction solution was prepared according to the following system: 10×Buffer 7 (40mM HEPES (6.8), 60mM KCl, 6mM MgCl2) 2μL, LbCas12a (1μM) 1μL, HopZ5 crRNA2 (10μM) 1μL, FB reporter (FAM-TTATT-Biotin, 10μM) 1μL, HopZ5 amplification product 1μL, H2O (RNAase free) was added to 20μL; an equal amount of RNase free H2O was added to the negative control; after adding the components of the rapid detection kit for the pathogenic bacteria of kiwifruit canker, the reaction was carried out at 37°C for 30min.

[0052] (2) Add 50 μL of PBS buffer to the above reaction solution, insert the lateral flow test strip into the PCR tube, observe the control line, remove the test strip after the control line appears, and observe the result with the naked eye within 5 minutes. If the detection band of the sample is obviously darker than the detection band of the negative control, the sample is judged to be positive, and take a photo to record. Figure 8 ).

[0053] Example 5 Natural sample test

[0054] This example is used to illustrate the performance of using a rapid detection kit for kiwifruit canker pathogens to detect random natural samples.

[0055] 1) 17 leaf samples were randomly collected from the Kiwifruit National Resource Garden of Wuhan Botanical Garden, and total DNA was extracted according to the method in Example 1;

[0056] 2) performing isothermal amplification on 17 samples according to the method in Example 1 to obtain HopZ5 amplification products;

[0057] 3) The detection reaction solution was prepared according to the following system: 10×Buffer 7 (40mM HEPES (6.8), 60mM KCl, 6mM MgCl2) 2μL, LbCas12a (1μM) 1μL, HopZ5 crRNA2 (10μM) 1μL, FQ reporter (FAM-TTATT-BHQ1, 10μM) 1μL, HopZ5 amplification product 1μL, H2O (RNAase free) was added to 20μL; after adding the components of the rapid detection kit for the pathogenic bacteria of kiwifruit canker, the reaction was carried out at 37°C for 30min.

[0058] 4) Use a portable fluorescent flashlight to illuminate the PCR tube and observe; two positive samples were identified among the 17 samples (1, 16), which is consistent with the cell culture test results ( Fig. 9 ).

Claims

1. A rapid detection kit for kiwifruit canker pathogens, characterized in that: It includes a HopZ5 specific primer, an LbCas12a protein, a HopZ5 crRNA, an FQ reporter molecule probe or an FB reporter molecule, and a buffer; the sequence of the HopZ5 crRNA is shown in SEQ ID NO: 1-6, including any one or more thereof.

2. The kit according to claim 1, characterized in that The sequences of the HopZ5 specific primers are shown in SEQ ID NOs: 7 and 8.

3. The kit according to claim 1, characterized in that The buffer solution comprises HEPS, KCl and MgCl2, and has a pH value of 6.8-8.

8.

4. The kit according to claim 3, characterized in that The buffer included 40 mM HEPES (6.8), 60 mM KCl, 6 mM MgCl2, and had a pH of 6.

8.

5. A method for detecting the pathogenic bacteria of kiwifruit canker based on the kit according to claim 1, characterized in that: After the LbCas12a protein specifically cuts the HopZ5 amplification product, it is detected by fluorescence or lateral flow test strips.

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