Method and kit for visualizing detection of alternaria alternata in wheat based on rpa-crispr / cas12a system

Abstract

The application discloses a method and a kit for visual detection of alternaria alternata based on an RPA-CRISPR / Cas12a system. The kit comprises specific RPA primer pairs and crRNA for detecting alternaria alternata. The alternaria alternata is detected by RPA amplification and crRNA recognition, and the infected plant or sample is detected as early as possible, and quarantine treatment is performed. The application has the characteristics of convenient operation, less time consumption, high sensitivity and strong specificity, and can effectively break away from the dependence on large instruments. The visual detection kit can quickly and accurately screen the samples containing alternaria alternata at quarantine ports, prevent the spread of the pathogen with seeds and other propagation materials, and has important significance for the detection, prevention and control of the plant pathogenic alternaria alternata and the promotion of the healthy development of agricultural production.

Description

[0001] Cross-references to related applications

[0002] This invention claims priority to Chinese Patent Application No. CN202411073321.4, filed on August 6, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biotechnology, specifically, it relates to a method and kit for visually detecting wheat scab based on the RPA-CRISPR / Cas12a system. Background Technology

[0004] Alternaria wheatii Alternaria triticina Prasada & Prabhu (also known as wheat leaf blight fungus) can cause wheat leaf blight, especially in central and eastern India, where it can damage wheat yields by 60-75% in severe cases. This pathogen is a seed-borne pathogen of wheat and durum wheat and can be spread through wheat seeds, agricultural machinery, and bulk feed grains, and may even be intentionally or unintentionally spread through passenger cargo.

[0005] Besides causing wheat leaf blight, *Alternaria alternata* can also infect various other plants such as oats, barley, rye, and sesame, seriously threatening crop production. Currently, with increasingly close international trade and the growing circulation of various agricultural products and commodities, the possibility of this pathogen entering and establishing itself in my country is high, warranting serious attention at entry ports. Therefore, establishing specific, sensitive, rapid, and effective molecular detection methods for the pathogen *Alternaria alternata*, and effectively identifying it, is of great significance for the rapid customs clearance of imported plants and plant products, early field disease diagnosis, and prevention of transmission.

[0006] Although the current entry-exit inspection and quarantine industry standard (SN / T 4733-2016) for the detection and identification of *Alternaria alternata* (wheat leaf blight pathogen) specifies the use of LJY1 / LJY2 specific primers for amplification, sequencing, and GenBank similarity (98-100%) comparison for identification, sequence comparison revealed that the species with the highest similarity between the LJY1 / LJY2 primer sequence and the sequence in GenBank is not *Alternaria alternata*, but other species. This may be because, on the one hand, there are many incorrectly identified *Alternaria* sequences in GenBank, making accurate species identification impossible using the method specified in the industry standard; on the other hand, the phylogenetic position of *Alternaria alternata* was not clear at the time the standard was formulated. Therefore, there is an urgent need to develop a rapid detection method for *Alternaria alternata* using accurately identified strains. Summary of the Invention

[0007] The application aims to provide a method and kit for visualizing detection of Alternaria alternata in wheat based on an RPA-CRISPR / Cas12a system.

[0008] To achieve the application purpose, in a first aspect, the application provides a crRNA for detecting Alternaria alternata in wheat, wherein a guide sequence of the crRNA is At-crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCGAUGCCACGGAGUAGUUCUAC-3' (SEQ ID NO: 3).

[0009] In a second aspect, the application provides an Alternaria alternata in wheat detection reaction system, comprising an RPA amplification reaction system and a Cas12a detection reaction system; the Cas12a detection reaction system comprises the crRNA as shown in SEQ ID NO: 3.

[0010] Further, the RPA amplification reaction system comprises an RPA primer pair as shown in SEQ ID NO: 1-2.

[0011] In a third aspect, the application provides a kit for visualizing detection of Alternaria alternata in wheat based on an RPA-CRISPR / Cas12a system, comprising the crRNA as shown in SEQ ID NO: 3 and the RPA primer pair as shown in SEQ ID NO: 1-2.

[0012] Further, the kit further comprises a Cas12a enzyme protein and an ssDNA reporter molecule.

[0013] The ssDNA reporter molecule is FQ-DNA or LF-DNA.

[0014] The reporter molecule FQ-DNA is 5'-(6-FAM) TTATT (BHQ1)-3' (fluorescence detection method).

[0015] The reporter molecule LF-DNA is 5'-(6-FAM) TTTTTTTTTT (Biotin)-3' (lateral flow test strip detection method).

[0016] In a fourth aspect, the application provides application of the kit in detection of Alternaria alternata in wheat.

[0017] In a fifth aspect, the application provides a method for visualizing detection of Alternaria alternata in wheat based on an RPA-CRISPR / Cas12a system, comprising the following steps:

[0018] S1. Extracting genomic DNA of a sample to be tested;

[0019] S2. Using the total DNA of step S1 as a template, performing RPA isothermal amplification reaction with the RPA primer pair shown in SEQ ID NO: 1-2 to obtain RPA amplification products;

[0020] S3. Using the RPA amplification products of step S2 as a template, adding a Cas12a detection reaction system to perform a CRISPR reaction;

[0021] The Cas12a detection reaction system comprises the crRNA, Cas12a enzyme protein and ssDNA reporter molecule;

[0022] The ssDNA reporter molecule is FQ-DNA or LF-DNA;

[0023] Reporter molecule FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3' (fluorescence detection method);

[0024] Reporter molecule LF-DNA: 5'-(6-FAM) TTTTTTTTTT (Biotin)-3' (lateral flow test strip detection method);

[0025] S4. Visualize the fluorescence reaction: the reaction product of step S3 with the reporter molecule FQ-DNA is subjected to color development under blue light (wavelength 470 nm) or naked eye observation. If the reaction product has no fluorescence brightness or naked eye observation has no brightness under blue light irradiation, it indicates that the sample to be tested does not contain wheat alternaria alternata. If the reaction product has fluorescence brightness or naked eye observation has brightness under blue light irradiation, it indicates that the sample to be tested contains wheat alternaria alternata.

[0026] Visual lateral flow test strip detection reaction: the reaction product obtained by adding the reporter molecule LF-DNA in step S3 is subjected to color development detection using a lateral flow test strip. If the test strip of the sample to be tested appears a red band or both the test strip of the sample to be tested and the negative sample control line position appear a red band, it indicates that the sample to be tested contains wheat alternaria alternata. If the test strip does not appear a red band, and a red band appears on the control line, it indicates that the sample to be tested does not contain wheat alternaria alternata.

[0027] Further, in the RPA isothermal amplification reaction system used in step S2, the concentrations of the forward primer and the reverse primer are both 480 nmon / L;

[0028] The amplification reaction conditions are: 35℃, 15-30 min (preferably 15 min).

[0029] Further, in the CRISPR reaction system used in step S3, the concentrations of crRNA, Cas12a enzyme protein and reporter molecule FQ-DNA are 100-300 nmon / L, 50-200 nmon / L and 50-800 nmon / L, respectively, and preferably 200 nmon / L, 100 nmon / L and 400 nmon / L.

[0030] The CRISPR reaction conditions are: 37℃, 15 min.

[0031] Further, in the CRISPR reaction system used in step S3, the concentrations of crRNA, Cas12a enzyme protein and reporter molecule FQ-DNA are 100-300 nmon / L, 50-200 nmon / L and 50-800 nmon / L, respectively, and preferably 200 nmon / L, 100 nmon / L and 400 nmon / L.

[0032] The CRISPR reaction conditions are: 37℃, 10-30 min (preferably 10 min).

[0033] By means of the above technical solution, the present application has at least the following advantages and beneficial effects:

[0034] (1) The visual detection kit for wheat Alternaria alternata provided by the present application can first amplify the sample to be detected by RPA, then guide the CRISPR-Cas12a system to recognize and cut the target double-stranded DNA under the mediation of the crRNA guide sequence to activate the non-specific nuclease function, then randomly cut the ssDNA reporter molecule in the system to obtain the cleavage product, and finally judge by colorimetric detection of the cleavage product. The RPA-CRISPR / Cas12a technology for detecting wheat Alternaria alternata provided by the present application can specifically and sensitively detect wheat Alternaria alternata through RPA amplification and crRNA recognition, with a detection limit as low as 10 pg, so that the plant or sample infected with wheat Alternaria alternata can be detected as early as possible for quarantine treatment. The present application has the characteristics of convenient operation, short time consumption, high sensitivity and strong specificity, and can be carried out in an environment of 35-37℃, so as to effectively break away from the dependence on large-scale laboratory instruments.

[0035] (2) The visual detection kit provided by the present application can quickly and accurately screen samples containing wheat Alternaria alternata at quarantine ports, prevent the spread of pathogens with seeds and other propagation materials, and has important significance for the detection, prevention and control of plant pathogenic wheat Alternaria alternata and the healthy development of agricultural production, and has great application and promotion value. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 A schematic diagram of the specificity of the RPA primer in the preferred embodiment of the present application;

[0037] Figure 2 A schematic diagram of the specificity of the primer and reporter molecule in the RPA combined with the CRISPR / Cas12a fluorescence detection method in the preferred embodiment of the present application;

[0038] Figure 3 A schematic diagram of the visualization result of the optimization of the concentration of the reporter molecule in the RPA combined with the CRISPR / Cas12a fluorescence detection method in the preferred embodiment of the present application;

[0039] Figure 4 A schematic diagram of the visualization result of the time optimization in the RPA combined with the CRISPR / Cas12a fluorescence detection method in the preferred embodiment of the present application;

[0040] Figure 5 A schematic diagram of the visualization result of the sensitivity detection in the RPA combined with the CRISPR / Cas12a fluorescence detection method in the preferred embodiment of the present application;

[0041] Figure 6 The specificity of the primer and reporter molecule in the RPA-CRISPR / Cas12a lateral flow test strip detection system in the preferred embodiment of the present application (T: test band; C: control line);

[0042] Figure 7 The detection result of the reporter molecule with different concentrations in the RPA-CRISPR / Cas12a lateral flow test strip detection system in the preferred embodiment of the present application (T: test band; C: control line);

[0043] Figure 8 Different reaction times in the RPA-CRISPR / Cas12a lateral flow test strip detection system in the preferred embodiment of the present application (T: test band; C: control line);

[0044] Figure 9 The sensitivity test of the RPA-CRISPR / Cas12a lateral flow test strip detection system in the preferred embodiment of the present application (T: test band; C: control line). DETAILED DESCRIPTION

[0045] The present application provides a primer, a probe, a detection method and a kit for visualizing detection of wheat alternaria alternata based on RPA amplification combined with a CRISPR / Cas12a system, which has the characteristics of easy operation, rapid sensitivity, accurate identification result, etc.

[0046] The present application adopts the following technical solutions:

[0047] The present application provides a primer, a probe, a detection method and a kit for visualizing detection of wheat alternaria alternata based on RPA amplification combined with a CRISPR / Cas12a system, which has the characteristics of easy operation, rapid sensitivity, accurate identification result, etc.Alternaria triticina ) type strain and glyceraldehyde-3-phosphate dehydrogenase of closely related fungi ( gapdh Gene sequences were analyzed and compared, and specific RPA amplification primer pairs and crRNA guide sequences were designed. The test sample was first subjected to RPA amplification, and then the CRISPR / Cas12a system was guided by the crRNA sequence to recognize and bind to the RPA amplification products and cut the target double-stranded DNA, activating the non-specific nuclease function. Then, the ssDNA reporter molecule in the system was randomly cut to obtain lysis products, and finally the lysis products were judged by colorimetric detection.

[0048] A kit for detecting wheat scab includes RPA enzyme lyophilized powder, RPA primers, crRNA guide sequence, Cas12a enzyme, and ssDNA reporter molecule;

[0049] The nucleotide sequences of the RPA primers are: At-RPA-F: 5'-CCCACTACGCTGTAAGCATCCCCGCGCGAAC-3' (SEQ ID NO:1), At-RPA-R: 5'-GCCTGCGTGTGTTAGCCTGCGTCCTGTAGCG-3' (SEQ ID NO:2), and the crRNA guide sequence is At-crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCGAUGCCACGGAGUAGUUCUAC-3' (SEQ ID NO:3).

[0050] The ssDNA reporter molecule is a single-stranded nucleotide sequence labeled with FAM at the 5' end and BHQ1 at the 3' end, or a single-stranded nucleotide sequence labeled with FAM at the 5' end and Biotin at the 3' end; the FAM and BHQ1 modified ssDNA reporter molecule FQ-DNA can be used to detect the presence of wheat spores in the target system with the naked eye under blue light (wavelength 470 nm) excitation, and the FAM and Biotin modified ssDNA reporter molecule LF-DNA is used for lateral flow test strip detection.

[0051] The nucleotide sequences of the ssDNA reporter molecules are FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3', LF-DNA: 5'-(6-FAM) TTTTTTTTTT (Biotin)-3'.

[0052] The application of the specific primer pair or the primer-probe combination includes any of the following:

[0053] (1) Identify whether the sample to be tested is *Alternaria alternata*;

[0054] (2) a kit for identifying Alternaria alternata in wheat;

[0055] (3) detecting whether the sample to be tested contains Alternaria alternata in wheat;

[0056] (4) a kit for detecting whether the sample to be tested contains Alternaria alternata in wheat.

[0057] The present application provides a kit for identifying Alternaria alternata in wheat, comprising the specific primer pair or the primer probe combination.

[0058] The kit further comprises one or more of Rehydration buffer, DEPC water, magnesium acetate, NEBuffer, lateral flow test strip and positive template.

[0059] The method for visualizing fluorescent detection or visualizing lateral flow test strip detection of Alternaria alternata comprises the following steps:

[0060] S1. Extracting the genomic DNA of the sample to be tested;

[0061] S2. RPA amplification: using the DNA of step S1 as a template, performing isothermal amplification reaction with the above-mentioned RPA primers to obtain RPA amplification products;

[0062] S3. CRISPR / Cas system reaction detection: using the above-mentioned crRNA guide sequence to prepare Cas12a / crRNA complex, and then adding ssDNA reporter molecule FQ-DNA (fluorescent detection method) or LF-DNA (lateral flow test strip detection method) and the RPA amplification products of step S2, performing cleavage reaction in the CRISPR / Cas12a system to obtain cleavage products;

[0063] S4. Visualizing fluorescent reaction: performing color development or naked eye observation of the cleavage products of step S3 under blue light (wavelength 470 nm), if the cleavage products have no fluorescence brightness or naked eye observation has no brightness under blue light irradiation, it indicates that the sample to be tested is not / does not contain Alternaria alternata in wheat, if the cleavage products have fluorescence brightness or naked eye observation has brightness under blue light irradiation, it indicates that the sample to be tested is / contains Alternaria alternata in wheat.

[0064] Visualizing lateral flow test strip detection reaction: using lateral flow test strip to perform color development detection of the cleavage products obtained in step S3, if the test band of the sample to be tested appears red band or the test band of the sample to be tested and the negative sample control line position all appear red band, it indicates that the sample to be tested is / contains Alternaria alternata in wheat, if the test band does not appear red band, and red band appears on the control line, it indicates that the sample to be tested is not / does not contain Alternaria alternata in wheat.

[0065] The system and conditions of the isothermal amplification reaction in step S2 are as follows: 50 μL system, that is, in a tube provided with RPA enzyme freeze-dried powder (TwistAmp Basic Kit of TwistDx Company), 29.5 μL of Rehydration buffer, 11.2 μL of DEPC-H2O, 2.4 μL of 10 μmol / L At-RPA-F primer, 2.4 μL of 10 μmol / L At-RPA-R primer, 2 μL of DNA, 2.5 μL of 280 mmol / L magnesium acetate, and after uniform mixing, the reaction tube is placed at 35 ℃, and heated for 15 min. TM The system and conditions of the isothermal amplification reaction in step S2 are as follows: 50 μL system, that is, in a tube provided with RPA enzyme freeze-dried powder (TwistAmp Basic Kit of TwistDx Company), 29.5 μL of Rehydration buffer, 11.2 μL of DEPC-H2O, 2.4 μL of 10 μmol / L At-RPA-F primer, 2.4 μL of 10 μmol / L At-RPA-R primer, 2 μL of DNA, 2.5 μL of 280 mmol / L magnesium acetate, and after uniform mixing, the reaction tube is placed at 35 ℃, and heated for 15 min.

[0066] The system and conditions of the lysis reaction of the fluorescence detection method in step S3 are as follows: 20 μL system, that is, 13.6 μL of DEPC-H2O, 2 μL of NEBuffer (10×), 0.4 μL of 5 μmol / L Cas12a enzyme protein, 1.6 μL of 5 μmol / L FQ-DNA, 0.4 μL of 0.01 mmol / L At-crRNA, and 2 μL of the RPA amplification product obtained in step S2, and after uniform mixing, the reaction tube is placed at 37 ℃, and heated for 15 min. Then color development observation is performed under blue light (wavelength 470 nm) (see step S4).

[0067] The system and conditions of the lysis reaction of the lateral flow test strip detection method in step S3 are as follows: 100 μL system, that is, 13.2 μL of DEPC-H2O, 2 μL of NEBuffer (10×), 0.4 μL of 5 μmol / L Cas12a enzyme protein, 2 μL of 5 μmol / L LF-DNA, 0.4 μL of 0.01 mmol / L At-crRNA, and 2 μL of the RPA amplification product obtained in step S2, and after uniform mixing, the reaction tube is placed at 37 ℃, and heated for 10 min. After the reaction is completed, 80 μL of DEPC-H2O is added, mixed uniformly, and then color development detection is performed using a lateral flow test strip (see step S4).

[0068] In the isothermal amplification reaction in step S3, the minimum detection sensitivity of the DNA is 10 pg.

[0069] In the constant temperature amplification reaction system of the fluorescence detection method in step S3, the final concentration of 400 nmol / L of the CRISPR / Cas12a reporter molecule FQ-DNA is the optimal choice, and the nucleotide sequence of the ssDNA reporter molecule is FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3'.

[0070] The final concentration of the CRISPR / Cas12a reporter molecule LF-DNA in the isothermal amplification reaction system of the lateral flow test strip detection method in step S3 is 100 nmol / L, which is the optimal choice, and the nucleotide sequence of the ssDNA reporter molecule is LF-DNA: 5'-(6-FAM) TTTTTTTTTT (Biotin)-3'.

[0071] In the isothermal amplification reaction in step S3, the concentration ratio of the Cas12a protein to the crRNA is 1:1.5-1:2.

[0072] In the present application, the sample to be tested can be a plant sample, a pure culture sample of fungi, a soil sample, etc., and the plant sample can be a leaf, a stem, a root, a fruit, etc.

[0073] The principle of the above-mentioned fluorescence detection method is that CRISPR / Cas12a recognizes a specific target in the RPA amplification product under the guidance of crRNA, and CRISPR / Cas12a, crRNA and target sequence molecules form a complex; the complex cuts the ssDNA reporter molecule FQ-DNA in the detection system, and a large amount of fluorescence is generated after the probe molecule is cut, which can be detected.

[0074] The principle of the above-mentioned lateral flow test strip detection method is that CRISPR / Cas12a recognizes a specific target in the RPA amplification product under the guidance of crRNA, and CRISPR / Cas12a, crRNA and target sequence molecules form a complex; the complex cuts the ssDNA reporter molecule LF-DNA in the detection system, and the product is added to the test strip after the reaction is completed, the probe molecule forms a conjugate with the gold particle avidin antibody, the probe molecule conjugate that is not cut is intercepted by the controlled streptavidin due to carrying biotin (Biotin), the cut probe molecule conjugate loses biotin (Biotin) and is not intercepted by the control line, and is intercepted by the fluorescent group (6-FAM) antigen in the subsequent test strip, thereby accumulating color development and completing detection.

[0075] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the technical means used in the examples is the conventional means familiar to those skilled in the art, and the raw materials used are commercially available goods.

[0076] Example 1 RPA primer design and amplification system determination

[0077] The sequences of wheat Alternaria alternata and its related fungi (Table 1) were obtained from the NCBI database gapdhGene sequences were sequenced, and sequence alignment and analysis were performed. Based on the specific target sequence of *Alternaria alternata*, two sets of candidate primer pairs that met the RPA primer design principles were designed. After a series of preliminary tests and screenings, the band brightness of the amplification products corresponding to each set of primers was detected by gel electrophoresis, and the following primers were finally determined:

[0078] Forward primer At-RPA-F sequence: 5'-CCCACTACGCTGTAAGCATCCCCGCGCGAAC-3' (SEQ ID NO:1)

[0079] Reverse primer At-RPA-R sequence: 5'-GCCTGCGTGTGTTAGCCTGCGTCCTGTAGCG-3' (SEQ ID NO:2)

[0080] The primer pairs that were not used are:

[0081] F3: 5'-CCCCGCGCGAACACAAACCCATAATAGCCCAC-3'

[0082] R3: 5'-GCCTGCGTGTGTTAGCCTGCGTCCTGTAGCG-3'

[0083] The amplified fragment size corresponding to primers At-RPA-F / At-RPA-R is 127 bp.

[0084] After determining the optimal primer pairs, an RPA amplification system was established, and the amplification temperature, amplification time, and other conditions were optimized.

[0085] The optimized RPA amplification system is as follows: In a tube containing lyophilized RPA enzyme powder (TwistDx TwistAmp...). TM Add 29.5 μL of rehydration buffer, 11.2 μL of DEPC-H2O, 2.4 μL of 10 μmol / L At-RPA-F, 2.4 μL of 10 μmol / L At-RPA-R, 2 μL of sample DNA, and 2.5 μL of 280 mmol / L magnesium acetate to the Basic Kit. Mix well and place the reaction tube at 35 °C for 15 min.

[0086] Gel electrophoresis results showed that the primer pair At-RPA-F / At-RPA-R had good specificity for Alternaria wheatis. Figure 1 ). Figure 1 Samples numbered 1-18 correspond to strains numbered 1-18 in Table 1. Only strain Alternaria wheati, number 1, showed an amplified band at 127bp.

[0087] Among the 9 strains of fungi shown in Table 1, the DNA of the strains whose strain numbers start with CBS is derived from the Westerdijk Fungal Biodiversity Centre in the Netherlands; the LC strains are isolated and identified by the inventors and preserved in the Cai Lei research group of the Institute of Microbiology, Chinese Academy of Sciences.

[0088] Table 1 Wheat Alternaria alternata and its closely related fungi for specific detection Alternaria = A. )

[0089]

[0090] Example 2 Establishment of RPA-CRISPR / Cas12a fluorescence detection system

[0091] 1. Among the 127 bp fragments amplified by RPA in Example 1, the crRNA guide sequence At-crRNA was designed by combining the PAM recognition site of CRISPR / Cas12a, and the sequence was 5'-UAAUUUCUACUAAGUGUAGAUCGAUGCCACGGAGUAGUUCUAC-3' (SEQ ID NO: 3).

[0092] The nucleotide sequence of the ssDNA reporter molecule is FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3'.

[0093] The above sequence is synthesized by Beijing Tianyihuiyuan Biotechnology Co., Ltd.

[0094] 2. The RPA-CRISPR / Cas12a fluorescence detection system uses the RPA enzyme and matching reagents of TwistDx TwistAmp TM Basic Kit, DEPC treated water is purchased from Shengong Bioengineering (Shanghai) Co., Ltd., and Cas12a is purchased from NEB Company in the United States.

[0095] 3. The detection method comprises the following steps

[0096] (1) Extracting the genomic DNA of the sample to be tested;

[0097] (2) Using the DNA template of step (1) and the RPA primers in Example 1 to perform isothermal amplification reaction to obtain RPA amplification product;

[0098] Isothermal amplification reaction system and conditions: in the tube containing RPA enzyme lyophilized powder (TwistDx TwistAmp TMAdd 29.5 μL of Rehydration buffer, 11.2 μL of DEPC-H2O, 2.4 μL of 10 μmol / L At-RPA-F primer, 2.4 μL of 10 μmol / L At-RPA-R primer, 2 μL of DNA, and 2.5 μL of 280 mmol / L magnesium acetate to the Basic Kit. Mix well and place the reaction tube at 35 °C for 15 min.

[0099] (3) Prepare a Cas12a / crRNA complex using the above At-crRNA guide sequence, then add ssDNA reporter molecule and RPA amplification product from step (2), and perform lysis reaction in CRISPR / Cas12a system to obtain lysis product;

[0100] The RPA-CRISPR / Cas12a amplification system consisted of: 14.4 μL DEPC-H2O, 2 μL NEBuffer (10×), 0.4 μL 5 μmol / L Cas12a enzyme protein, 0.8-1.6 μL 5 μmol / L FQ-DNA (preferably 1.6 μL), 0.4 μL 0.01 mmol / L LAt-crRNA, and 2 μL of the RPA amplification product from Example 1. After mixing thoroughly, the reaction tube was placed at 37 °C and heated for 15-30 min (preferably 15 min). The reaction was then observed under blue light (wavelength 470 nm).

[0101] (4) The lysis products of step (3) are subjected to color development or naked-eye observation under blue light (wavelength 470 nm). If the lysis products have no fluorescence under blue light irradiation or no brightness under naked-eye observation, it indicates that the sample to be tested is not / does not contain Alternaria wheatis. If the lysis products have fluorescence under blue light irradiation or brightness under naked-eye observation, it indicates that the sample to be tested is / contains Alternaria wheatis.

[0102] Test results as follows Figure 2 As shown in the figure, the PCR tubes from left to right represent the detection results of bacterial species numbered 1-18 in Table 1. It can be seen from the figure that only the amplification product of Alternaria wheati, number 1, shows a clear fluorescent signal.

[0103] Example 3: Optimization and Sensitivity Detection of the RPA-CRISPR / Cas12a Fluorescence Detection System

[0104] The RPA-CRISPR / Cas12a fluorescence detection system established in Example 2 was further optimized to make the entire reaction faster and more efficient, while reducing costs.

[0105] 1. Optimization of the concentration of fluorescent reporter molecule FQ-DNA

[0106] The concentration of the fluorescent reporter molecule FQ-DNA was optimized, and the reporter molecule concentrations were set to 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, and 800 nmol / L, with other conditions unchanged. The RPA-CRISPR / Cas12a fluorescent detection system established in Example 2 was used to determine the concentration of the fluorescent reporter molecule FQ-DNA.

[0107] The detection results of different concentrations of fluorescent reporter molecules are shown in Figure 3 The PCR tubes from left to right represent reporter molecule concentrations of 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, and 800 nmol / L, respectively.

[0108] The results show that the higher the concentration of the CRISPR / Cas12a fluorescent reporter molecule, the stronger the fluorescence intensity. When the final concentration of the reporter molecule is 100 nmol / L or more, the fluorescence signal can be observed. When the final concentration of the reporter molecule is 800 nmol / L, the fluorescence intensity is extremely obvious. To balance the cost and fluorescence intensity, the final concentration of the CRISPR / Cas12a fluorescent reporter molecule is 400 nmol / L, which is the optimal choice.

[0109] 2. Isothermal amplification reaction time optimization

[0110] The RPA-CRISPR / Cas12a fluorescent detection system established in Example 2 was used, and the RPA reaction time was unchanged at 15 min. The reaction time of step (3) in Example 2 was set to 5 min, 10 min, 15 min, 20 min, and 30 min, respectively, with other conditions unchanged. Sterile water was used as a negative control.

[0111] The detection results of different reaction times are shown in Figure 4 The PCR tubes from left to right represent reaction times of 5 min, 10 min, 15 min, 20 min, and 30 min, respectively.

[0112] The results show that the longer the reaction time, the stronger the fluorescence intensity. When the reaction time reaches 5 min, the fluorescence can be detected. As the CRISPR reaction time increases, the fluorescence intensity increases. When the reaction time is 15 min, the fluorescence intensity is obvious. Therefore, 15 min can be selected as the CRISPR / Cas12a fluorescent detection reaction time.

[0113] 3. Sensitivity experiment

[0114] The RPA-CRISPR / Cas12a fluorescence detection system established in Example 2 was used, wherein the concentration of the fluorescence reporter molecule FQ-DNA and the isothermal amplification reaction time were adjusted according to the results of the optimization in Example 3, the template DNA concentration of the target species was diluted by 10 times, and 6 concentrations were set, i.e. 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, and 0.01 pg, and 3 replicates were set for each concentration gradient.

[0115] The sensitivity detection results are shown in FIG. 3. Figure 5 The PCR tubes from left to right represent the fluorescence reporter molecule concentrations of 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, and 0.01 pg, respectively.

[0116] The results show that the fluorescence signal can be generated when the concentration of the wheat Alternaria alternata genomic DNA is higher than 10 pg. It is shown that the sensitivity of the CRISPR / Cas12a fluorescence detection method is 10 pg.

[0117] In summary, the total reaction time is within 30 min (RPA reaction for 15 min and cleavage for 15 min), and the sensitivity of the wheat Alternaria alternata RPA-CRISPR / Cas12a fluorescence detection is 10 pg.

[0118] Example 4: Establishment of RPA-CRISPR / Cas12a lateral flow test strip detection system

[0119] 1. In the 127 bp fragment amplified by RPA in Example 1, a crRNA guide sequence At-crRNA was designed in combination with the CRISPR / Cas12a recognition site PAM, and the sequence was 5'-UAAUUUCUACUAAGUGUAGAUCGAUGCCACGGAGUAGUUCUAC-3'.

[0120] The nucleotide sequence of the ssDNA reporter molecule is LF-DNA: 5'-(6-FAM) TTTTTTTTTT (Biotin)-3'.

[0121] The above sequence was synthesized by Beijing Tianyihuiyuan Biotechnology Co., Ltd.

[0122] 2. The RPA-CRISPR / Cas12a lateral flow test strip detection system uses the RPA enzyme and matching reagent of TwistAmp TM BasicKit of TwistDx Company, the RPA enzyme and matching reagent of TwistAmp BasicKit are purchased from Shengong Biotechnology Co., Ltd. (Shanghai), the Cas12a enzyme protein is purchased from NEB Company in the United States, and DEPC treated water is purchased from Shengong Biotechnology Co., Ltd. (Shanghai).

[0123] 3. The detection method comprises the following steps:

[0124] (1) Extract genomic DNA from the sample to be tested;

[0125] (2) Using the DNA template from step (1), an isothermal amplification reaction was performed using the RPA primers from Example 1 to obtain the RPA amplification product;

[0126] Isothermal amplification reaction system and conditions: In tubes containing lyophilized RPA enzyme powder (TwistDx, TwistAmp). TM Add 29.5 μL of Rehydration buffer, 11.2 μL of DEPC-H2O, 2.4 μL of 10 μmol / L At-RPA-F primer, 2.4 μL of 10 μmol / L At-RPA-R primer, 2 μL of DNA, and 2.5 μL of 280 mmol / L magnesium acetate to the Basic Kit. Mix well and place the reaction tube at 35 °C for 15 min.

[0127] (3) Prepare a Cas12a / crRNA complex using the above At-crRNA guide sequence, then add ssDNA reporter molecule and RPA amplification product from step (2), and perform lysis reaction in CRISPR / Cas12a system to obtain lysis product;

[0128] Isothermal amplification reaction system and conditions: DEPC-H2O 13.2 μL, NEBuffer (10×) 2 μL, 5 μmol / L Cas12a enzyme protein 0.4 μL, 5 μmol / L LF-DNA 2 μL, 0.01 mmol / L At-crRNA 0.4 μL, and 2 μL of RPA amplification product obtained in step S2. After mixing thoroughly, the reaction tube was placed at 37 ℃ and heated for 10–30 min. After the reaction was complete, 80 μL of DEPC-H2O was added and mixed thoroughly.

[0129] (4) The lysis products obtained in step S3 are detected by color development using a lateral flow test strip. If a red band appears on the test strip of the sample to be tested or a red band appears on both the test strip of the sample to be tested and the control line of the negative sample, it indicates that the sample to be tested is / contains Alternaria wheatis. If no red band appears on the test strip but a red band appears on the control line, it indicates that the sample to be tested is / does not contain Alternaria wheatis.

[0130] Test results as follows Figure 6 As shown in the figure, the test strips from left to right represent the detection results of bacterial species numbered 1-18 in Table 1. It can be seen from the figure that only the amplification product of Alternaria wheati (serial number 1) shows a detection band.

[0131] Example 5 Optimization of RPA-CRISPR / Cas12a lateral flow test strip detection system and sensitivity detection

[0132] Further optimize the RPA-CRISPR / Cas12a lateral flow test strip detection system established in Example 4 to make the whole reaction more rapid and efficient, while reducing the cost.

[0133] 1. Optimization of lateral flow test strip reporter molecule LF-DNA concentration

[0134] The concentration of lateral flow test strip reporter molecule LF-DNA was optimized, and the reporter molecule concentration was set to 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, 800 nmol / L, and other conditions were unchanged. Sterile water was used as a negative control. The RPA-CRISPR / Cas12a lateral flow test strip detection system established in Example 4 was used to determine the concentration of test strip reporter molecule LF-DNA.

[0135] The detection results of test strip reporter molecules with different concentrations are shown in Figure 7 . From left to right, the test strip represents reporter molecule concentrations of 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, and 800 nmol / L, respectively.

[0136] The results show that when the reporter molecule concentration in the 100 μL test strip detection system is 50 nmol / L, the test band (T line) is obvious.

[0137] 2. Isothermal amplification reaction time optimization

[0138] The RPA-CRISPR / Cas12a lateral flow test strip detection system established in Example 4 was used, and the RPA reaction time was unchanged, i.e. 15 min. The reaction time of step (3) in Example 4 was set to 5 min, 10 min, 15 min, 20 min, and 30 min, respectively, and other conditions were unchanged. Sterile water was used as a negative control.

[0139] The detection results of different reaction times are shown in Figure 8 . From left to right, the test strip represents amplification reaction times of 5 min, 10 min, 15 min, 20 min, and 30 min, respectively.

[0140] The results show that detectable products can be produced after 10 min of reaction. Therefore, 10 min is selected as the preferred reaction time for CRISPR / Cas12a.

[0141] 3. Sensitivity experiment

[0142] The RPA-CRISPR / Cas12a lateral flow test strip detection system established in Example 4 was used, wherein the concentration of the reporter molecule LF-DNA and the isothermal amplification reaction time were adjusted according to the results of the optimization in Example 5, the template DNA concentration of the target species was diluted by 10 times, and a total of 6 concentrations were set, i.e. 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, and 0.01 pg, and 3 replicates were set for each concentration gradient.

[0143] The sensitivity detection results are shown in FIG. 6. Figure 9 The test strips from left to right represent 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, and 0.01 pg, respectively.

[0144] The results show that the concentration of the wheat Alternaria alternata genomic DNA is 10 pg, which can produce a weak detection signal, and the detection signal is enhanced with the increase of the concentration.

[0145] In summary, the total reaction time is within 25 min (RPA reaction for 15 min and cleavage for 10 min), and the sensitivity of the wheat Alternaria alternata RPA-CRISPR / Cas12a lateral flow test strip is 10 pg.

[0146] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A reaction system for detecting wheat Alternaria, characterized in that, It includes an RPA amplification reaction system and a Cas12a detection reaction system; the Cas12a detection reaction system includes crRNA for detecting wheat scab. The guide sequence of the crRNA is At-crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCGAUGCCACGGAGUAGUUCUAC-3'; The RPA amplification reaction system includes RPA primer pairs with sequences as shown in SEQ ID NO:1-2.

2. A kit for visual detection of wheat scab based on the RPA-CRISPR / Cas12a system, characterized in that, It includes the crRNA as described in claim 1 and the RPA primer pair as shown in SEQ ID NO:1-2.

3. The reagent kit according to claim 2, characterized in that, The kit also includes Cas12a enzyme protein and ssDNA reporter molecule; The ssDNA reporter molecule is either FQ-DNA or LF-DNA; Reporter molecule FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3', based on fluorescence detection method; The reporter molecule is LF-DNA; 5'-(6-FAM)TTTTTTTTTT (Biotin)-3', based on the lateral flow test strip detection method.

4. The application of the kit described in claim 2 or 3 in the detection of wheat Alternaria.

5. A method for visually detecting wheat scab based on the RPA-CRISPR / Cas12a system, characterized in that, Includes the following steps: S1. Extract genomic DNA from the sample to be tested; S2. Using the total DNA from step S1 as a template, perform an RPA isothermal amplification reaction using the RPA primer pair shown in SEQ ID NO:1-2 to obtain the RPA amplification product; S3. Using the RPA amplification product obtained in step S2 as a template, add it to the Cas12a detection reaction system to carry out a CRISPR reaction; The Cas12a detection reaction system includes the crRNA, Cas12a enzyme protein, and ssDNA reporter molecule as described in claim 1; The ssDNA reporter molecule is either FQ-DNA or LF-DNA; Reporter molecule FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3', based on fluorescence detection method; Reporter molecule LF-DNA: 5'-(6-FAM)TTTTTTTTTT (Biotin)-3', based on lateral flow test strip detection method; S4. Visualizing the fluorescence reaction: The reaction product of adding the reporter molecule FQ-DNA in step S3 is subjected to color development under blue light at a wavelength of 470 nm or observed with the naked eye. If the reaction product has no fluorescence brightness under blue light irradiation or no brightness when observed with the naked eye, it indicates that the sample to be tested does not contain Alternaria wheati. If the reaction product has fluorescence brightness under blue light irradiation or brightness when observed with the naked eye, it indicates that the sample to be tested contains Alternaria wheati. Visualized lateral flow test strip detection reaction: The reaction product obtained by adding the reporter molecule LF-DNA in step S3 is detected by color development using a lateral flow test strip. If a red band appears on the test band of the sample to be tested, or if red bands appear on both the test band of the sample to be tested and the control line of the negative sample, it indicates that the sample to be tested contains Alternaria wheati. If no red band appears on the test band, but a red band appears on the control line, it indicates that the sample to be tested does not contain Alternaria wheati.

6. The method according to claim 5, characterized in that, In step S2, the concentrations of both the forward and reverse primers in the RPA isothermal amplification reaction system were 480 nmon / L. The amplification reaction conditions were: 35℃, 15-30 min.

7. The method according to claim 6, characterized in that, The amplification reaction conditions for step S2 are: 35℃, 15 min.

8. The method according to claim 5, characterized in that, In step S3, the concentrations of crRNA, Cas12a enzyme protein, and reporter molecule FQ-DNA in the CRISPR reaction system were 100-300 nmon / L, 50-200 nmon / L, and 50-800 nmon / L, respectively; the CRISPR reaction conditions were 37℃ for 15-30 min.

9. The method according to claim 8, characterized in that, In the CRISPR reaction system used in step S3, the concentrations of crRNA, Cas12a enzyme protein, and reporter molecule FQ-DNA were 200 nmon / L, 100 nmon / L, and 400 nmon / L, respectively; the CRISPR reaction conditions were 37℃ for 15 min.

10. The method according to claim 5, characterized in that, In the CRISPR reaction system used in step S3, the concentrations of crRNA, Cas12a enzyme protein, and ssDNA reporter molecule LF-DNA were 100-300 nmon / L, 50-200 nmon / L, and 50-800 nmon / L, respectively. The CRISPR reaction conditions are: 37℃, 10-30 min.

11. The method according to claim 10, characterized in that, In the CRISPR reaction system used in step S3, the concentrations of crRNA, Cas12a enzyme protein, and ssDNA reporter molecule LF-DNA were 200 nmol / L, 100 nmol / L, and 100 nmol / L, respectively. The CRISPR reaction conditions were: 37°C for 10 min.

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