Rpa-cr ispr / cas12a-based visual detection of pandoraea pulmonalis test kit and detection method

Abstract

The present application relates to the technical field of biotechnology and plant disease detection, in particular, the present application relates to a visual detection kit and detection method for pantoea ananatis based on RPA-CRISPR / Cas12a.The present application constructs specific RPA-CRISPR / Cas12a detection primers for pantoea ananatis through research and screening, and combines the method with an immunolateral flow test paper, the detection result can be directly reflected by means of nucleic acid test paper, which has the advantages of strong specificity, high sensitivity, short detection time, visualization, etc., and is not limited by materials, site and instrument, and can directly detect the target pathogenic bacteria from the disease sample, providing a simple and easy-to-use detection method for planters and primary plant protection workers.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and plant disease detection technology, and in particular, to a detection kit and method for visually detecting Pantotheca pineapple based on RPA-CRISPR / Cas12a. Background Technology

[0002] Pantoea ananatis is an important plant pathogenic bacterium that can cause bacterial diseases in many major and economic crops, including rice, corn, onions, strawberries, melons, tea, pineapples, and sugarcane, causing serious damage. Because bacterial diseases develop rapidly and control measures are limited, it is necessary to develop advanced rapid detection methods to ensure timely detection at the early stages of disease occurrence and to implement control measures to curb its spread.

[0003] Currently, molecular detection techniques for *U. pineapple pantothecin* are mainly based on PCR. However, PCR-based molecular detection requires specialized instruments such as PCR machines, gel imaging systems, and quantitative real-time PCR instruments, and is costly, making it unsuitable for rapid field detection. Recombinase polymerase amplification technology, involving multiple enzymes, exponentially amplifies nucleic acids under isothermal conditions, and is considered a potential alternative to PCR. This technology not only eliminates the need for high-value instruments but also offers a shorter reaction time. Clustered regularly spaced short palindromic repeats and CRISPR-related proteins, collectively known as the CRISPR / Cas system, is a novel nucleic acid detection technology that has been widely used in recent years for detecting various pathogens. It can combine fluorescence detection and lateral flow strip detection methods to differentiate the results. Since fluorescence detection still requires high-value fluorescence detection instruments, developing a method system suitable for lateral flow strip detection is a crucial requirement for the rapid detection of *U. pineapple pantothecin*. Summary of the Invention

[0004] Based on the need for rapid, field-applicable detection of *U. pineapple*, this invention has developed a visualization-based detection kit and method for *U. pineapple* based on RPA-CRISPR / Cas12a through in-depth research. Specifically, the technical solution of this invention is as follows:

[0005] In one aspect, the present invention discloses a reagent for visual detection of *Ureaplasma pineapple* based on RPA-CRISPR / Cas12a, the reagent comprising primer pairs RPA-F and RPA-R for detecting *Ureaplasma pineapple*, 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, the present invention provides a kit for visual detection of Ureaplasma pineapple based on RPA-CRISPR / Cas12a, which comprises the above-mentioned reagents.

[0007] In one embodiment, the kit further includes Cas12a and an LF-DNA reporter molecule.

[0008] In one aspect, the present invention also provides the application of a reagent or kit for visually detecting Pantotheca pineapple based on RPA-CRISPR / Cas12a in the detection of Pantotheca pineapple.

[0009] In one aspect, this invention discloses a visual detection method for *Ureaplasma pineapple* based on RPA-CRISPR / Cas12a, the method comprising the following steps:

[0010] (1) Extract genomic DNA from the sample to be tested;

[0011] (2) Mix the primer pairs RPA-F and RPA-R for detecting Pantotheca pineapple with the DNA obtained in step (1) and perform RPA amplification to obtain the RPA amplification product.

[0012] (3) Mix the RPA amplification product described in step (2) with crRNA, Cas12a, and LF-DNA reporter molecules, perform a CRISPR reaction, and read the detection signal;

[0013] (4) Determine whether the sample to be tested contains Pantotheca pineapple based on 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 / Cas12a reaction time is 20 min; the final concentration of the reporter molecule added in the CRISPR / Cas12a reaction is determined to be 100 nmol / L.

[0016] In this invention, the determination of whether a sample contains *Ureaplasma pineapple* is based on the bands on the test strip: if only one red band appears in the control area, the result is negative, indicating that the sample does not contain *Ureaplasma pineapple*; if red bands appear in both the detection and control areas, or only in the detection area, the result is positive, indicating that the sample contains *Ureaplasma pineapple*; if there is no band in the control area, the result is invalid. Beneficial effects

[0017] This solution proposes a rapid and visualized detection system for Pantotheca pineapple based on RPA-CRISPR / Cas12a technology. The reaction conditions are simple, the detection speed is fast, and the detection results can be intuitively reflected by nucleic acid test strips. It has the advantages of high specificity, high sensitivity, short detection time, and visualization. It is not limited by materials, site, and instruments, and can directly detect the target pathogen from diseased samples, providing growers and grassroots plant protection workers with a simple and easy-to-use detection method. Attached Figure Description

[0018] Figure 1 The figures above illustrate the results of optimizing the cutting time using CRISPR / Cas12a in this embodiment of the scheme. From left to right, they are 30, 25, 20, 15, 10 min and blank control.

[0019] Figure 2 The figures above are schematic diagrams of the test results for optimizing the addition ratio of RPA products in CRISPR / Cas12a in this embodiment of the scheme, from left to right: 2, 4, 6, 8, 10, 12 μL.

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

[0021] Figure 4 The figures 1 and 2 are schematic diagrams illustrating the sensitivity of the RPA-CRISPR / Cas12a detection system in this embodiment, from left to right representing 1 and 1×10⁻⁶, respectively. –1 1×10 –2 1×10 –3 1×10 –4 ng / μL of Pantotheca pineapple genomic DNA and blank control (water).

[0022] Figure 5 The diagram above illustrates the results of the RPA-CRISPR / Cas12a detection system in this embodiment, from left to right: Pantoea 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 Compared with the blank control (water). Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0024] 1.1 Primer Design

[0025] Based on the reference genome of Pantotheca pineapple in the NCBI database, and considering that RPA-CRISPR / Cas12a detection technology requires specific primers to simultaneously meet the requirements of RPA reaction and the requirements of the PAM sequence (TTTN or NAAA) of the CRISPR / Cas12a recognition site, and that the 20 bases immediately following the PAM sequence are preferably specific, this invention designed primer pairs and crRNA sequences as shown in Table 1.

[0026] Table 1. Primer Information Table

[0027] 1.2 DNA extraction from strains

[0028] Pantotheca pineapple was inoculated onto LB medium and cultured in a shaker at 28°C for 15 hours. The bacterial cells were collected by centrifugation, and genomic DNA was extracted using a commercially available bacterial genome extraction kit. The DNA content and purity were determined using Nanodrop.

[0029] 1.3 RPA Amplification System

[0030] The RPA amplification kit purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd. was used. The amplification system according to the instructions was 50 μL: 29.4 μL of A Buffer, 2 μL each of forward and reverse primers (10 μmol / L), 2 μL of DNA template, and 12.1 μL of dd H2O were added to the dry powder reaction tube. 2.5 μL of B Buffer was added to the tube cap. After mixing well, the reaction tube was placed at 42℃ for 30 min.

[0031] 1.4 CRISPR / Cas12a Detection System

[0032] The CRISPR / Cas12a test strip detection system consists of 20 μL: 2 μL of 10×NE Buffer r2.1, 1 μmol / L LabCas12a (Cpf1) 1 μL, 10 μmol / L crRNA 1 μL, 10 μmol / L LF-DNA 1 μL, 0.5 μL of 40 U / μL RNase inhibitor, 10.5 μL of nuclease-free water, and 4 μL of RPA reaction product. After mixing the reaction system thoroughly, incubate at 37°C for 20 min. After the reaction is complete, add 80 μL of deionized water. Insert the binding pad end of the Cas12 / 13 nucleic acid test strip into the reaction tube and interpret the results. Under natural light, if a red band appears on the control line and the test line does not develop color, the result is negative. If a visible red band appears on the test line, the result is positive (the presence or absence of a band on the control line is acceptable). If there is no band on the control line, the result is invalid.

[0033] 2.1 Optimization of CRISPR / Cas12a Cutting Time

[0034] CRISPR cutting reaction times were set to 10, 15, 20, 25, and 30 min, and test strips were used for detection, with three replicates for each group. The optimal cutting time was determined based on whether the test strip detection line developed color at different reaction times. Results showed that the test strip detection line color deepened with increasing CRISPR / Cas12a cutting time (see [link to relevant documentation]). Figure 1 When the CRISPR / Cas12a reaction time was 20 min, the detection lines on the test strip were very obvious. Therefore, the CRISPR / Cas12a cutting time was optimized to 20 min.

[0035] 2.2 Optimization of RPA product ratio in RPA-CRISPR / Cas12a reaction

[0036] The volumes of RPA product added to the CRISPR / Cas12a reaction system were set to 2, 4, 6, 8, 10, and 12 μL, and test strips were used for detection, with three replicates for each experiment. The optimal RPA product addition ratio was determined based on whether the test strip showed color and the intensity of the band. Results (see...) Figure 2 The results showed that when the RPA reaction product added to CRISPR / Cas12a was 2-6 μL, the bands became more and more obvious as the added volume increased. When the RPA product volume was more than 6 μL, the test strip detection lines were all very obvious but there was no significant difference. Therefore, the volume of RPA product added to the CRISPR / Cas12a reaction was optimized to 6 μL.

[0037] 2.3 Optimization of the reporter molecule ratio in the RPA-CRISPR / Cas12a reaction

[0038] The final concentrations of LF-DNA reporter molecules added to the CRISPR / Cas12a reaction system were set to 50, 100, 150, 200, and 250 nmol / L, and test strips were used for detection, with three replicates for each experiment. The optimal ratio of reporter molecules was determined based on whether the test strip showed color and the intensity of the band. Results (see...) Figure 3 The results showed that the color of the test strip's detection line deepened with increasing reporter molecule concentration. When the final concentration of the reporter molecule added to CRISPR / Cas12a was greater than 100 nmol / L, the test strip's detection lines were all clearly visible but without significant difference. Therefore, the final concentration of the reporter molecule added to the CRISPR / Cas12a reaction was determined to be 100 nmol / L.

[0039] 2.4 Sensitivity evaluation of the RPA-CRISPR / Cas12a detection system

[0040] At concentrations of 1 and 1×10, respectively –1 1×10 –2 1×10 –3 1×10 –4 RPA amplification was performed using ng / μL of *Ustilago maydis* genomic DNA as a template, and detection was performed using an optimized CRISPR / Cas12a system, with dd H2O as a control. The experiment was performed in triplicate. The limit of detection (LOD) was determined by whether the test line on the test strip showed color. Results (see [link]). Figure 4 This indicates that 1 ng / μL to 1×10 –3 All ng / μL test strips showed positive results, 1×10 –4 No bands were observed on the test strips in the ng / μL test group, indicating a negative result. This demonstrates that the detection limit of the Pantotheca pineapple assay system is 1×10⁻⁶. –3 It has a high sensitivity of ng / μL.

[0041] 2.5 Specificity Validation of the RPA-CRISPR / Cas12a Detection System

[0042] DNA extracted from all tested strains (see Table 2) was used as templates in an optimized RPA-CRISPR / Cas12a system for detection, with dd H2O as a control. Triple replicates were performed. Specificity of the system was determined by whether the test line on the test strip developed color. Test strip results (see Table 2) Figure 5 The results showed that only the test strip for Pantotheca pineapple was positive, while the rest were negative, indicating that the system established in this invention can specifically detect Pantotheca pineapple.

[0043] Table 2. Strains used for specificity verification in this embodiment.

[0044] Note: + indicates the presence of a specific test band; - indicates the absence of a test band.

[0045] In summary, this invention establishes an optimized CRISPR / Cas12a system to verify specific target sequences after RPA-specific amplification using a test strip method. The detection results can be intuitively reflected using nucleic acid test strips. It has advantages such as simple operation, high specificity, high sensitivity, short detection time, and visualization. Moreover, it is not limited by materials, site, or instruments, providing growers and grassroots plant protection workers with a simple and easy-to-use rapid detection method for pathogen identification.

[0046] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A visual detection method for *Ureaplasma pineapple* based on RPA-CRISPR / Cas12a, characterized in that, The method includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Mix the primer pairs RPA-F and RPA-R for detecting Pantotheca pineapple with the DNA obtained in step (1) and perform RPA amplification to obtain the RPA amplification product. (3) Mix the RPA amplification product described in step (2) with crRNA, Cas12a, and LF-DNA reporter molecules, perform a CRISPR / Cas12a reaction, and read the detection signal; (4) Determine whether the sample to be tested contains Pantotheca pineapple based on the detection signal; 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; the nucleotide sequence of crRNA is shown in SEQ ID NO.3; the CRISPR / 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.

2. The detection method according to claim 1, characterized in that, The determination of whether a sample contains Pantothenia bromelain is based on the bands on the test strip. If only one red band appears in the control area, the result is negative, indicating that the sample does not contain Pantothenia bromelain. If red bands appear in both the test area and the control area, the result is positive, indicating that the sample contains Pantothenia bromelain. If the charge sheet is blank, the charge is deemed invalid.

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