Detection kit and detection method for visually detecting pantoea ananatis based on RPA-CRISPR / Cas12a

Abstract

The invention relates to the technical field of biotechnology and plant disease detection, in particular to a detection kit and a detection method for visual detection of pantoea ananatis based on RPA-CRISPR / Cas12a. A specific RPA-CRISPR / Cas12a detection primer aiming at pantoea ananatis is constructed through research and screening, the method is combined with immune lateral flow test paper, a detection result can be visually reflected by means of a nucleic acid test strip, and the method has the advantages of being high in specificity and sensitivity, short in detection time, visual and the like, is not limited by materials, sites and instruments, and can be widely applied to detection of pantoea ananatis. Target pathogenic bacteria can be directly detected from disease samples, and a simple and easy detection method is provided for growers and grass-roots plant protection workers.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology and plant disease detection, in particular, the present application relates to a RPA-CRISPR / Cas12a-based visual detection kit and detection method for Pantoea ananatis. BACKGROUND

[0002] Pantoea ananatis is an important plant pathogenic bacterium that can cause bacterial diseases in rice, corn, onion, strawberry, melon, tea tree, pineapple, sugarcane and many other staple crops and economic crops, and cause serious damage. Because bacterial diseases develop rapidly and there are limited control measures, it is necessary to develop advanced rapid detection methods in order to detect them in time at the beginning of the disease and take control measures to curb the development of the disease.

[0003] At present, the molecular detection technology for Pantoea ananatis is mainly based on PCR, and the PCR-based molecular detection technology requires special instruments such as PCR instrument, gel imaging instrument, and fluorescence quantitative PCR instrument, and the detection cost is high, which is not suitable for rapid detection in the field. Recombinase polymerase amplification technology (RPA) is an exponential amplification of nucleic acids under isothermal conditions with the participation of multiple enzymes, which is called a nucleic acid detection technology that can replace PCR. This technology not only eliminates the need for high-value instruments, but also has a short reaction time. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR / Cas system) is a new type of nucleic acid detection technology that has been widely used for pathogen detection in recent years. It can combine fluorescence detection and lateral flow test strip detection to determine the detection results. Since the fluorescence detection method still requires high-value fluorescence detection instruments, the development of a method system suitable for lateral flow test strip detection has become an important requirement for rapid detection of Pantoea ananatis. SUMMARY

[0004] Based on the need for rapid detection of Pantoea ananatis in the field, the present application develops a RPA-CRISPR / Cas12a-based visual detection kit and detection method for Pantoea ananatis through in-depth research. Specifically, the technical scheme of the present application is as follows:

[0005] In one aspect of the present application, a RPA-CRISPR / Cas12a-based visual detection reagent for Pantoea ananatis is disclosed, which comprises a primer pair RPA-F, RPA-R for detecting Pantoea ananatis, and crRNA; wherein the nucleotide sequence of RPA-F is shown in SEQ ID NO. 1; the nucleotide sequence of RPA-R is shown in SEQ ID NO. 2; and the nucleotide sequence of crRNA is shown in SEQ ID NO. 3.

[0006] In one aspect of the present application, the present application provides a RPA-CRISPR / Cas12a-based kit for visual detection of Pantoea ananatis, which comprises the above-mentioned reagent.

[0007] In one embodiment, the kit further comprises Cas12a and LF-DNA reporter molecules.

[0008] In one aspect of the present application, the present application also provides the use of a RPA-CRISPR / Cas12a-based reagent or kit for visual detection of Pantoea ananatis in detecting Pantoea ananatis.

[0009] In one aspect of the present application, the present application discloses a RPA-CRISPR / Cas12a-based method for visual detection of Pantoea ananatis, which comprises the following steps:

[0010] (1) extracting genomic DNA of the sample to be tested;

[0011] (2) mixing the primer pair RPA-F and RPA-R for detecting Pantoea ananatis with the DNA obtained in step (1) to perform RPA amplification, thereby obtaining an RPA amplification product;

[0012] (3) mixing the RPA amplification product of step (2) with crRNA, Cas12a and LF-DNA reporter molecules to perform a CRISPR reaction, and reading the detection signal;

[0013] (4) determining whether the sample to be tested contains Pantoea ananatis according to the detection signal;

[0014] The nucleotide sequence of RPA-F is shown in SEQ ID NO. 1; the nucleotide sequence of RPA-R is shown in SEQ ID NO. 2; and the nucleotide sequence of crRNA is shown in SEQ ID NO. 3.

[0015] In a preferred embodiment, the CRISPR R / Cas12a reaction time is 20 min; and the final concentration of the reporter molecule added in the CRISPR / Cas12a reaction is determined to be 100 nmol / L.

[0016] In the present application, determining whether the sample to be tested contains Pantoea ananatis according to the detection signal is determined according to the strip of the test strip: if the test strip only has a red strip in the quality control area, the result is negative, indicating that the sample to be tested does not contain Pantoea ananatis; if the test strip has a red strip in the detection area and the quality control area, or only the detection area has a red strip, the result is positive, indicating that the sample to be tested contains Pantoea ananatis; and if there is no strip in the quality control area, it is determined to be invalid. Advantages

[0017] The scheme proposes a pineapple P. pentosaceum visual rapid detection system based on RPA-CRISPR / Cas12a technology. The reaction condition is simple, the detection speed is fast, the detection result can be directly reflected by the nucleic acid test strip, and the system has the advantages of strong specificity, high sensitivity, short detection time, visualization, etc. It is not limited by materials, site and instrument, and can directly detect target pathogenic bacteria from disease samples. It provides a simple and easy detection method for planters and primary plant protection workers. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Fig. 1 is a schematic diagram of the results of optimizing the cutting time of CRISPR / Cas12a in the embodiment of the scheme. From left to right, 30, 25, 20, 15, 10 min and blank control;

[0019] Figure 2 Fig. 4 is a schematic diagram of the test results of optimizing the RPA product addition ratio of CRISPR / Cas12a in the embodiment of the scheme. From left to right, 2, 4, 6, 8, 10, 12 μL;

[0020] Figure 3 Fig. 5 is a schematic diagram of the results of optimizing the reporter molecule concentration of CRISPR / Cas12a in the embodiment of the scheme. From left to right, the final concentration is 250, 200, 150, 100, 50 nmol / L and blank control;

[0021] Figure 4 Fig. 6 is a schematic diagram of the results of the sensitivity of the RPA-CRISPR / Cas12a detection system in the embodiment of the scheme. From left to right, 1, 1×10 –1 , 1×10 –2 , 1×10 –3 , 1×10 –4 ng / μL of P. pentosaceum genomic DNA and blank control (water).

[0022] Figure 5Pantoea ananatis; P. agglomerans, P. stewartii, P. eucrina, Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, X. campestris pv. campestris, Enterobacteriaceae roggenkampii, Dickeya zeae, Burkholderia gladioli and blank control (water) from left to right. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with examples. The equipment and reagents used in each example and test example can be obtained from commercial channels unless otherwise specified. The reagents used in the present application are all analytical grade reagents unless otherwise specified. The specific examples described herein are only used to explain the present application and do not limit the present application.

[0024] Example 1: Establishment of a method for visual detection of Pantoea ananatis based on RPA-CRISPR / Cas12a

[0025] 1.1 Primer design

[0026] Based on the Pantoea ananatis reference genome in the NCBI database, and the requirements of RPA reaction and CRISPR / Cas12a recognition site PAM sequence (TTTN or NAAA) when designing specific primers, the 20 bases after the PAM sequence are preferably specific, and considering the above factors, the primer pairs and crRNA sequences shown in Table 1 are designed.

[0027] Table 1. Primer information table

[0028] 1.2 Extraction of bacterial DNA

[0029] Pantoea ananatis was inoculated into LB medium and cultured at 28°C for 15h on a shaker. The bacterial cells were collected by centrifugation, and the genomic DNA was extracted using a commercially available bacterial genomic extraction kit. The DNA content and purity were determined by Nanodrop.

[0030] 1.3 RPA amplification system

[0031] RPA amplification kit purchased from Amply Future (Changzhou) Biotechnology Co., Ltd. was used, and the amplification system according to the instructions was 50 μL: 29.4 μL of A Buffer, 2 μL of each of the upstream and downstream primers (10 μmol / L), 2 μL of DNA template, 12.1 μL of dd H2O added to the dry powder reaction tube, 2.5 μL of B Buffer added to the tube cover, and the reaction tube was placed at 42°C for 30 min after mixing evenly.

[0032] 1.4 CRISPR / Cas12a detection system

[0033] The CRISPR / Cas12a test strip detection system was 20 μL: 2 μL of 10x NE Buffer r2.1, 1 μL of 1 μmol / L LabCas12a (Cpf1), 1 μL of 10 μmol / L crRNA, 1 μL of 10 μmol / L LF-DNA, 0.5 μL of 40 U / μL RNase inhibitor, 10.5 μL of nuclease-free water, and 4 μL of RPA reaction product. The reaction system was mixed evenly and reacted at 37°C for 20 min. After the reaction was completed, 80 μL of deionized water was added, the binding pad end of the Cas12 / 13 special nucleic acid test strip was inserted into the reaction tube, and the result was interpreted. Under natural light, if the quality control line appears a red band, the detection line does not develop color, and it is determined to be a negative result. If the detection line appears a red band visible to the naked eye, it is determined to be a positive result (whether the quality control line has a band or not), and if the quality control line has no band, it is determined to be invalid.

[0034] Example 2: Optimization of RPA-CRISPR / Cas12a detection system

[0035] 2.1 Optimization of CRISPR / Cas12a cleavage time

[0036] The CRISPR cleavage reaction time was set to 10, 15, 20, 25 and 30 min, and the test strip detection was performed, with 3 repeats for each experiment. According to whether the test strip detection line developed color under different reaction times, the optimal cleavage time was selected. The results showed that as the CRISPR / Cas12a cleavage time increased, the color of the test strip detection line deepened (see Figure 1 ). When the CRISPR / Cas12a reaction time was 20 min, the test strip detection line band was very obvious, so the CRISPR / Cas12a cleavage time was optimized to 20 min.

[0037] 2.2 Optimization of RPA product ratio in RPA-CRISPR / Cas12a reaction

[0038] The volume of RPA product added in the CRISPR / Cas12a reaction system was set to 2, 4, 6, 8, 10, 12 μL, and test strip detection was performed, with 3 repeats in each group of experiments. According to whether the test strip detection line developed color and the strength of the strip, the optimal RPA product addition ratio was selected. The results (see Figure 2 ) showed that when the RPA reaction product added in CRISPR / Cas12a was 2-6 μL, the strip band became more and more obvious as the volume increased, and when the volume of RPA product was more than 6 μL, the test strip detection line strip was very obvious but had no obvious difference, so the volume of RPA product added in the CRISPR / Cas12a reaction was optimized to 6 μL.

[0039] 2.3 Optimization of reporter molecule ratio in RPA-CRISPR / Cas12a reaction

[0040] The final concentration of LF-DNA reporter molecule added in the CRISPR / Cas12a reaction system was set to 50, 100, 150, 200, 250 nmol / L, and test strip detection was performed, with 3 repeats in each group of experiments. According to whether the test strip detection line developed color and the strength of the strip, the optimal reporter molecule addition ratio was selected. The results (see Figure 3 ) showed that as the concentration of the reporter molecule increased, the color of the test strip detection line deepened. When the final concentration of the reporter molecule added in CRISPR / Cas12a was greater than 100 nmol / L, the test strip detection line strip was very obvious but had no obvious difference, so the final concentration of the reporter molecule added in the CRISPR / Cas12a reaction was determined to be 100 nmol / L.

[0041] 2.4 Sensitivity evaluation of RPA-CRISPR / Cas12a detection system

[0042] P. ananatis genomic DNA with concentrations of 1, 1×10 –1 , 1×10 –2 , 1×10 –3 , 1×10 –4 ng / μL was used as a template for RPA amplification, and the optimized CRISPR / Cas12a system was used for detection, with dd H2O as a control, and 3 repeats were set for the test. According to whether the test strip detection line developed color, the detection lower limit of the system was determined. The results (see Figure 4 ) showed that the test strip detection results were positive from 1 ng / μL to 1×10 –3 ng / μL, and the test strip detection line of the 1×10 –4 ng / μL test group was not observed to have a strip, which was a negative result, indicating that the detection lower limit of the P. ananatis detection system was 1×10–3 ng / μL, with higher sensitivity.

[0043] 2.5 Specificity verification of RPA-CRISPR / Cas12a detection system

[0044] The extracted DNA of all test strains (see Table 2) was used as a template for detection by the RPA-CRISPR / Cas12a optimization system, with dd H2O as a control, and three replicates were set up. Whether the test strip detection line showed coloration was used to determine whether the system had specificity. The test strip results (see Table 3) showed that only the test strip of Pantoea ananatis was positive, and the rest were negative, indicating that the system established in the present application can specifically detect Pantoea ananatis. Figure 5

[0045] Table 2 Strains used for specificity verification in this embodiment , Note: + indicates specific test strip; - indicates no test strip.

[0046] In summary, after RPA specific amplification, the optimized CRISPR / Cas12a system is used to verify the specific target sequence by test strip method, and the detection result can be directly reflected by the nucleic acid test strip, which has the advantages of simple operation, strong specificity, high sensitivity, short detection time, visualization, etc., and is not limited by materials, site and instruments, providing a simple and easy pathogenic identification rapid detection method for growers and primary plant protection workers.

[0047] The above content is a further detailed description of the present application in combination with a specific embodiment, and cannot be considered as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, without departing from the concept of the present application, a number of simple deductions or substitutions can be made, which should be considered as belonging to the protection scope determined by the claims submitted by the present application.​

Claims

1. A reagent for visual detection of Pantoea pineapple based on RPA-CRISPR / Cas12a, characterized in that The reagent includes a primer pair RPA-F and RPA-R for detecting Pantoea ananas, and crRNA; wherein the nucleotide sequence of RPA-F is shown as SEQ ID NO.1; the nucleotide sequence of RPA-R is shown as SEQ ID NO.2; and the nucleotide sequence of crRNA is shown as SEQ ID NO.

3.

2. A kit for visual detection of Pantoea pineapple based on RPA-CRISPR / Cas12a, characterized in that The kit comprises the reagent according to claim 1.

3. The kit according to claim 2, wherein The kit also includes Cas12a and LF-DNA reporter molecules.

4. Use of the reagent according to claim 1 or the kit according to claim 2 in detecting Pantoea pineapple.

5. A method for visually detecting Pantoea pineapple based on RPA-CRISPR / Cas12a, characterized in that: The method comprises the following steps: (1) Extracting genomic DNA from the sample to be tested; (2) mixing the primer pair RPA-F and RPA-R for detecting Pantoea ananas with the DNA obtained in step (1), performing RPA amplification to obtain an RPA amplification product; (3) Mix the RPA amplification product described in step (2) with crRNA, Cas12a, and LF-DNA reporter molecules, perform CRISPRR / Cas12a reaction, and read the detection signal; (4) determining whether the sample to be tested contains Pantoea ananas based on the detection signal; Among them, the nucleotide sequence of RPA-F is shown in SEQ ID NO.1; the nucleotide sequence of RPA-R is shown in SEQ ID NO.2; and the nucleotide sequence of crRNA is shown in SEQ ID NO.

3.

6. The detection method according to claim 5, characterized in that The CRISPRR / Cas12a reaction time was 20 min; the final concentration of the reporter molecule added to the CRISPR / Cas12a reaction was determined to be 100 nmol / L.

7. The detection method according to claim 5, characterized in that Determining whether the sample to be tested contains Pantoea ananas according to the detection signal is based on the strips on the test strip. If only a red strip appears in the quality control area of ​​the test strip, the result is negative, indicating that the sample to be tested does not contain Pantoea ananas; if red strips appear in both the detection area and the quality control area of ​​the test strip, or only the detection area, the result is positive, indicating that the sample to be tested contains Pantoea ananas. If there is no band in the quality control area, the judgment is invalid.

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