Method and kit for visually detecting brassicaceous vegetable phytophthora parasitica var nicotianae

Abstract

The invention discloses a method and a kit for visually detecting phytophthora parasitica var nicotianae of brassicaceous vegetables, and particularly relates to a method and a kit for visually detecting phytophthora parasitica var nicotianae based on an RPA-CRISPR / Cas12a system. A specific RPA primer pair and crRNA for detecting the brassicaceous vegetable phytophthora parasitica var nicotianae are designed according to the difference of ITS sequences of the brassicaceous vegetable phytophthora parasitica var nicotianae and the sibling species of the brassicaceous vegetable phytophthora parasitica var nicotianae. The brassicaceous vegetable phytophthora parasitica var nicotianae is detected through double specificity of RPA amplification and crRNA recognition, the sensitivity of a fluorescence detection method and the sensitivity of a lateral flow test strip detection method are both 10 pg, and the detection sensitivity can be further improved by increasing the RPA amplification reaction time. The method has the characteristics of convenience in operation, low time consumption, high sensitivity, strong specificity and the like, and the whole process is carried out in an environment of 35-37 DEG C. The visual detection kit provided by the invention can be used for rapidly and accurately screening a sample containing the brassicaceous vegetable phytophthora parasitica var nicotianae.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method and a kit for visual detection of brassica black stem and leaf diseases. BACKGROUND

[0002] Brassica black stem and leaf diseases Plenodomus lingam (Synonym: Leptosphaeria maculans ), also known as Leptosphaeria maculans, is a plant quarantine pathogenic fungus in the List of Invasive Plant Quarantine Pests of the People's Republic of China. Its pathogenicity is very strong, and it seriously threatens the safety of rape production. The stem and leaf diseases of rape caused by it can cause economic losses of up to 1.5 billion US dollars. Brassica black stem and leaf diseases are widely distributed in many countries in Africa, America, Europe, Oceania and Asia, and have not been reported in China. In addition to rape, the pathogen can also infect brassica plants such as Chinese cabbage, cabbage, radish and wild radish, causing whole seedlings and adult plants to die.

[0003] Brassica black stem and leaf diseases can be transmitted by seeds or diseased residues, and there have been interception records at ports such as Shanghai, Xiamen, Chongqing and Nanjing in China. Its risk of entering China is increasing. Therefore, it is of great significance to establish a specific, sensitive, rapid and effective molecular detection method for the pathogen Brassica black stem and leaf diseases, to effectively identify Brassica black stem and leaf diseases, and to inspect and quarantine imported plants and plant products, diagnose early diseases in the field and block transmission.

[0004] The rapid detection techniques for X. campest ris currently include PCR amplification (CN 101805793B, Liu et al. 2006), LAMP (Du et al. 2021), LAMP-LFD (CN 104818339 A), RPA (Lei et al. 2019), etc. However, the PCR amplification method not only takes a long time, but also needs experimental equipment such as PCR instrument, gel electrophoresis and imaging system, which has certain limitations and is not suitable for on-site detection. The LAMP method also has obvious shortcomings, i.e. the probability of false positives is high and false detection is easy to occur. Although the RPA method can complete the reaction under constant temperature conditions, it still needs to be used with a fluorescence quantitative PCR instrument. Recently, Lei et al. (2022) developed a CRISPR / Cas12a convenient detection method for X. campestris, but we found that the reverse primer sequence used by them has one base difference with the corresponding sequence of the model strain of the pathogen, and the guide RNA sequence has one base difference with the corresponding sequence of most reported strains of the pathogen, which is likely to cause inefficient detection of X. campestris and the possibility of missed detection. In view of this, we redesigned the primer and guide RNA sequence of the pathogen and optimized the detection method. SUMMARY

[0005] The purpose of the present application is to provide a method and kit for visual detection of X. campestris (based on RPA-CRISPR / Cas12a system).

[0006] To achieve the purpose of the present application, in the first aspect, the present application provides a crRNA for detecting X. campestris Plenodomus lingam The guide sequence of the crRNA is Pi-crRNA: 5'-UAAUUUCUACUCUUGUAGAUGGGGAUCCAAUUGGUGGGCAAG-3' (SEQ ID NO: 3).

[0007] In the second aspect, the present application provides a X. campestris detection reaction system, which comprises a RPA amplification reaction system and a Cas12a detection reaction system; the Cas12a detection reaction system comprises a crRNA as shown in SEQ ID NO: 3.

[0008] Further, the RPA amplification reaction system comprises a RPA primer pair as shown in SEQ ID NO: 1-2.

[0009] Pi-RPA-F: 5'-CCGCCTCGATCAGTGGCGGCAGTCTACTTTGAT-3' (SEQ ID NO: 1) Pi-RPA-R: 5'-AAGGCGAGTCCCAAGTGGAACAAACACCCAACAC-3' (SEQ ID NO: 2) In a third aspect, the present application provides a kit for visualizing detection of Phytophthora brassicae based on RPA-CRISPR / Cas12a system, comprising a crRNA as shown in SEQ ID NO: 3 and a RPA primer pair as shown in SEQ ID NO: 1-2.

[0010] Further, the kit further comprises RPA enzyme, RPA buffer (such as: Rehydration buffer), magnesium acetate, NEBuffer, Cas12a enzyme protein, ssDNA reporter molecule.

[0011] The ssDNA reporter molecule is FQ-DNA or LF-DNA; Reporter molecule FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3' (fluorescence detection method); Reporter molecule LF-DNA; 5'-(6-FAM) TTTTTTTTTT (Biotin)-3' (lateral flow test strip detection method).

[0012] In a fourth aspect, the present application provides the use of the kit in the detection of Phytophthora brassicae.

[0013] In a fifth aspect, the present application provides a method for visualizing detection of Phytophthora brassicae based on RPA-CRISPR / Cas12a system, comprising the following steps: S1. Extracting the genomic DNA of the sample to be tested; S2. Using the total DNA of step S1 as a template, performing RPA isothermal amplification reaction with the RPA primer pair as shown in SEQ ID NO: 1-2 to obtain RPA amplification product; S3. Using the RPA amplification product obtained in step S2 as a template, adding a Cas12a detection reaction system to perform CRISPR reaction; The Cas12a detection reaction system comprises the crRNA, Cas12a enzyme protein and ssDNA reporter molecule; The ssDNA reporter molecule is FQ-DNA or LF-DNA; Reporter molecule FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3' (fluorescence detection method); Reporter molecule LF-DNA; 5'-(6-FAM) TTTTTTTTTT (Biotin)-3' (lateral flow test strip detection method); S4. Visualizing the fluorescence reaction: the reaction product in step S3 with the reporter molecule FQ-DNA is color-developed under blue light (wavelength 470 nm) or observed with naked eyes, if the reaction product has no fluorescence brightness under blue light or no brightness under naked eyes, it indicates that the sample to be tested does not contain the Brassica vegetable black stem rot fungus, if the reaction product has fluorescence brightness under blue light or brightness under naked eyes, it indicates that the sample to be tested contains the Brassica vegetable black stem rot fungus; Visualizing the lateral flow test strip detection reaction: the reaction product obtained by adding the reporter molecule LF-DNA in step S3 is color-developed and detected using a lateral flow test strip, if the test strip of the sample to be tested appears a red band or the test strip of the sample to be tested and the negative sample control line position both appear a red band, it indicates that the sample to be tested contains the Brassica vegetable black stem rot fungus, 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 the Brassica vegetable black stem rot fungus.

[0014] 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; The amplification reaction conditions are: 35℃, 15-30 min.

[0015] Further, in the CRISPR reaction system used in step S3, the concentrations of the crRNA, the Cas12a enzyme protein and the reporter molecule FQ-DNA are 100-300 nmon / L, 50-200 nmon / L and 50-800 nmon / L respectively, and are preferably 100 nmon / L, 50 nmon / L and 200 nmon / L respectively.

[0016] The CRISPR reaction conditions are: 37℃, 15-30 min.

[0017] Further, in the CRISPR reaction system used in step S3, the concentrations of the crRNA, the Cas12a enzyme protein and the reporter molecule LF-DNA are 100-300 nmon / L, 50-200 nmon / L and 50-800 nmon / L respectively, and are preferably 100 nmon / L, 50 nmon / L and 100 nmon / L respectively.

[0018] The CRISPR reaction conditions are: 37℃, 15-30 min.

[0019] By means of the above technical solutions, the present application has at least the following advantages and beneficial effects: (I) The kit for visually detecting the brassica black stem pathogen provided by the present application can first amplify the DNA of the sample to be tested through RPA, then under the mediation of the crRNA guide sequence, guide the CRISPR-Cas12a system to recognize and cut the target double-stranded DNA amplification product to activate the non-specific nuclease function, then cut the ssDNA reporter molecule in the system to obtain the cleavage product, and finally judge through the colorimetric detection of the cleavage product. The RPA-CRISPR / Cas12a technology for detecting the brassica black stem pathogen in the present application can specifically detect the brassica black stem pathogen through RPA amplification and crRNA recognition, has high specificity and high sensitivity (the DNA detection limit can be as low as 10 pg when the experimental cost and the shortest detection time are controlled), can detect the plant or sample infected by the brassica black stem pathogen as early as possible, and can be treated for quarantine. 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 DEG C, so as to effectively get rid of the dependence on large laboratory instruments.

[0020] (II) The visual detection kit provided by the present application can quickly and accurately screen the samples containing the brassica black stem pathogen at the quarantine port, prevent the spread of the pathogen with the import and export of seeds and other propagation materials, and has important significance for the detection, prevention and control of the plant pathogenic brassica black stem pathogen and the promotion of the healthy development of agricultural production, and has great application and popularization value. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a specific result diagram of the RPA primer specificity in the preferred embodiment of the present application.

[0022] Figure 2 It is a specific result diagram of the primer and reporter molecule specificity of the RPA combined CRISPR / Cas12a fluorescence detection method in the preferred embodiment of the present application.

[0023] Figure 3 It is a visual result diagram of the reporter molecule concentration optimization of the RPA combined CRISPR / Cas12a fluorescence detection method in the preferred embodiment of the present application.

[0024] Figure 4 It is a visual result diagram of the time optimization of the RPA combined CRISPR / Cas12a fluorescence detection method in the preferred embodiment of the present application.

[0025] Figure 5 It is a visual result diagram of the sensitivity detection of the RPA combined CRISPR / Cas12a fluorescence detection method in the preferred embodiment of the present application.

[0026] Figure 6The specificity of primers and reporter molecules in the RPA-CRISPR / Cas12a lateral flow test strip detection system in a preferred embodiment of the present invention is shown in the figure (T: test band; C: control line).

[0027] Figure 7 The results of different concentrations of reporter molecules in the RPA-CRISPR / Cas12a lateral flow test strip detection system in a preferred embodiment of the present invention are shown (T: test band; C: control line).

[0028] Figure 8 Different reaction times (T: test strip; C: control line) in the RPA-CRISPR / Cas12a lateral flow test strip detection system in a preferred embodiment of the present invention.

[0029] Figure 9 Sensitivity test of the RPA-CRISPR / Cas12a lateral flow test strip detection system in a preferred embodiment of the present invention (T: test strip; C: control line). Detailed Implementation

[0030] This invention provides primers, detection methods, and kits for visual detection of blackleg fungus in cruciferous vegetables based on RPA amplification combined with the CRISPR / Cas12a system. These methods are easy to operate, rapid, sensitive, and provide accurate identification results.

[0031] The present invention adopts the following technical solution: This invention addresses the blackleg fungus affecting cruciferous vegetables (… Plenodomus lingam The ITS sequences of the model strain and closely related fungi 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 product 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 the lysis product, and finally the colorimetric detection of the lysis product was used for judgment.

[0032] This invention provides a kit for detecting blackleg fungus in cruciferous vegetables, comprising RPA primers, crRNA guide sequence, ssDNA reporter molecule, RPA enzyme lyophilized powder, RPA buffer (rehydration buffer), magnesium acetate, NE buffer, and Cas12a enzyme; The nucleotide sequence of the RPA primer is: Pi-RPA-F: 5'-CCGCCTCGATCAGTGGCGGCAGTCTACTTTGAT-3' (SEQ ID NO: 1), Pi-RPA-R: 5'-AAGGCGAGTCCCAAGTGGAACAAACACCCAACAC-3' (SEQ ID NO: 2), and the crRNA guide sequence is Pi-crRNA: 5'-UAAUUUCUACUCUUGUAGAUGGGGAUCCAAUUGGUGGGCAAG-3' (SEQ ID NO: 3).

[0033] 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; wherein the FAM and BHQ1 modified ssDNA reporter molecule FQ-DNA can be used to visually detect the presence or absence of Alternaria brassicae in the target system 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.

[0034] The nucleotide sequence of the ssDNA reporter molecule is FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3', and LF-DNA: 5'-(6-FAM) TTTTTTTTTT (Biotin)-3'.

[0035] The application of the specific primer pair or the primer probe combination includes any one of the following: (1) identifying whether the sample to be tested is Alternaria brassicae; (2) preparing a kit for identifying Alternaria brassicae; (3) detecting whether the sample to be tested contains Alternaria brassicae; (4) preparing a kit for detecting whether the sample to be tested contains Alternaria brassicae.

[0036] The kit further comprises one or more of DEPC water or ddH2O, a PCR tube, a lateral flow test strip, and a positive template.

[0037] The application also provides a method for visually detecting or visually detecting Alternaria brassicae by lateral flow test strip, comprising the following steps: S1. Extracting the genomic DNA of the sample to be tested; S2. RPA amplification: using the DNA of step S1 as a template, performing isothermal amplification reaction by using the above RPA primer to obtain the RPA amplification product; S3. CRISPR / Cas system reaction detection: prepare Cas12a / crRNA complex using the above-mentioned crRNA guide sequence, then add ssDNA reporter molecule FQ-DNA (fluorescence detection method) or LF-DNA (lateral flow test strip detection method) and RPA amplification product of step S2, carry out cleavage reaction in the CRISPR / Cas12a system to obtain cleavage product; S4. Visual fluorescence reaction: develop or naked eye observe the cleavage product of step S3 under blue light (wavelength 470 nm), if the cleavage product has 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 cruciferous vegetable black stem rot fungus, if the cleavage product has fluorescence brightness or naked eye observation has brightness under blue light irradiation, it indicates that the sample to be tested is / is contains cruciferous vegetable black stem rot fungus.

[0038] Visual lateral flow test strip detection reaction: develop the cleavage product obtained in step S3 using a lateral flow test strip, if the test strip of the sample to be tested appears a red band or the test strip of the sample to be tested and the negative sample control line position both appear a red band, it indicates that the sample to be tested is / is contains cruciferous vegetable black stem rot fungus, 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 is not / does not contain cruciferous vegetable black stem rot fungus.

[0039] The system and conditions of the isothermal amplification reaction in step S2 are as follows: 50 μL system, that is, in the tube containing RPA enzyme freeze-dried powder (TwistAmp Basic Kit from TwistDx company), add RPA buffer (Rehydration buffer) 29.5 μL, DEPC-H2O or ddH2O 11.2 μL, 10 μmol / L Pi-RPA-F primer 2.4 μL, 10 μmol / L Pi-RPA-R primer 2.4 μL, 2 μL DNA, 280 mmol / L magnesium acetate 2.5 μL, mix uniformly, then place the reaction tube at 35 ℃ and heat for 15 min. TM Basic Kit) adding RPA buffer (Rehydration buffer) 29.5 μL, DEPC-H2O or ddH2O 11.2 μL, 10 μmol / L Pi-RPA-F primer 2.4 μL, 10 μmol / L Pi-RPA-R primer 2.4 μL, 2 μL DNA, 280 mmol / L magnesium acetate 2.5 μL, mix uniformly, then place the reaction tube at 35 ℃ and heat for 15 min.

[0040] The lysis reaction system and conditions of the fluorescence detection method in step S3 are as follows: 20 μL system, DEPC-H2O or ddH2O 14.8 μL, NEBuffer (10x) 2 μL, 5 μmol / L Cas12a enzyme protein (NEB M0653T EnGen Lba Cas12a (Cpf1)) 0.2 μL, 5 μmol / L FQ-DNA 0.8 μL, 0.01 mmol / L Pi-crRNA 0.2 μL, 2 μL of the RPA amplification product obtained in step S2, and after mixing uniformly, the reaction tube is placed at 37 ℃ and heated for 20 min. Then color development observation is carried out under blue light (wavelength 470 nm) (see step S4).

[0041] The lysis reaction system and conditions of the lateral flow test strip detection method in step S3 are as follows: 100 μL system, DEPC-H2O or ddH2O 11.6 μL, NEBuffer (10x) 2 μL, 5 μmol / L Cas12a enzyme protein 0.2 μL, 5 μmol / L LF-DNA 4 μL, 0.01 mmol / L Pi-crRNA 0.2 μL, 2 μL of the RPA amplification product obtained in step S2, and after mixing uniformly, the reaction tube is placed at 37 ℃ and heated for 20 min. After the reaction is completed, 80 μL of DEPC-H2O is added and mixed uniformly, and then color development detection is carried out using a lateral flow test strip (see step S4).

[0042] In the isothermal amplification reaction in step S3, the minimum detection sensitivity of the DNA is 10 pg.

[0043] In the isothermal amplification reaction system of the fluorescence detection method in step S3, the final concentration of 200 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'.

[0044] In the isothermal amplification reaction system of the lateral flow test strip detection method in step S3, the final concentration of 100 nmol / L of the CRISPR / Cas12a reporter molecule LF-DNA is the optimal choice, and the nucleotide sequence of the ssDNA reporter molecule is LF-DNA: 5'-(6-FAM) TTTTTTTTTT (Biotin)-3'.

[0045] In the isothermal amplification reaction in step S3, the concentration ratio of the Cas12a enzyme protein to the crRNA is 1:2.

[0046] 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.

[0047] 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 the target sequence molecule combine into 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.

[0048] The principle of the above-mentioned lateral flow test strip detection method is as follows: CRISPR / Cas12a recognizes a specific target in the RPA amplification product under the guidance of crRNA, and CRISPR / Cas12a, crRNA and the target sequence molecule combine into 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 and the gold particle anti-biotin antibody form a conjugate, the conjugate of the probe molecule that is not cut is intercepted by the controlled streptavidin due to carrying biotin (Biotin), and the conjugate of the probe molecule that is cut is not intercepted by the control line due to losing biotin (Biotin), and is intercepted by the fluorescent group (6-FAM) antigen in the subsequent test strip, so that the color development is completed and the detection is completed.

[0049] 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 are conventional means familiar to those skilled in the art, and the raw materials used are commercially available.

[0050] Example 1: RPA primer design and amplification system determination The ITS sequences of Brassicaceae vegetable black stem rot fungi and their related species fungi (Brassicaceae black stem rot fungi: KJ574223, JX499035, KT225526, AJ550887, JF740235, JF740234, FJ172239, DQ458907, MW810259, GU205260, AJ550889; see Fig. 33 of the literature Persoonia 47, 2021: 45-105 for the ITS sequence number of related species) were obtained from the NCBI database, and sequence alignment and analysis were performed. Two groups of candidate primer pairs meeting the RPA primer design principles were designed according to the specific target sequences of Brassicaceae vegetable black stem rot fungi. After a series of preliminary detection and screening, the amplification product bands corresponding to each group of primers were detected by gel electrophoresis, and the following primers were finally determined according to the band brightness: Forward primer Pi-RPA-F sequence: 5'-CCGCCTCGATCAGTGGCGGCAGTCTACTTTGAT-3' (SEQ ID NO: 1) Reverse primer Pi-RPA-R sequence: 5'-AAGGCGAGTCCCAAGTGGAACAAACACCCAACAC-3' (SEQ ID NO: 2) The amplified fragment size corresponding to primers Pi-RPA-F / Pi-RPA-R is 357 bp.

[0051] After determining the optimal primer pairs, an RPA amplification system was established, and the amplification temperature, amplification time, and other conditions were optimized.

[0052] The optimized RPA amplification system is as follows: In a tube containing lyophilized RPA enzyme powder (TwistDx TwistAmp...). TM Add 29.5 μL of RPA buffer (Rehydration buffer), 11.2 μL of DEPC-H2O or ddH2O, 2.4 μL of 10 μmol / L Pi-RPA-F, 2.4 μL of 10 μmol / L Pi-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.

[0053] Gel electrophoresis results showed that the primer pair Pi-RPA-F / Pi-RPA-R had good specificity for blackleg fungus, the causal agent of cruciferous vegetables. Figure 1 ). Figure 1 Samples numbered 1-13 correspond to strains numbered 1-13 in Table 1. The blackleg fungus strains of cruciferous vegetables numbered 7, 8, and 9 show an amplified band at 357 bp.

[0054] Of the 13 fungi shown in Table 1, the DNA of strains with strain numbers beginning with CBS originated from the Westerdijk Culture Collection in the Netherlands; the LC strain was isolated and identified by the inventor and is deposited in the Cai Lei research group at the Institute of Microbiology, Chinese Academy of Sciences.

[0055] Table 1. *Blackleg fungus* and its closely related fungi used for specific detection in cruciferous vegetables (… Plenodomus = P. )

[0056] Example 2: Establishment of the RPA-CRISPR / Cas12a fluorescence detection system 1. In the 357 bp fragment amplified by RPA in Example 1, the PAM recognition site of CRISPR / Cas12a is combined to design the crRNA guide sequence Pi-crRNA, and the sequence is 5'-UAAUUUCUACUCUUGUAGAUGGGGAUCCAAUUGGUGGGCAAG-3' (SEQ ID NO: 3).

[0057] The nucleotide sequence of the ssDNA reporter molecule is FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3'.

[0058] The above sequence is synthesized by Beijing Tianyihuiyuan Biotechnology Co., Ltd.

[0059] 2. The RPA-CRISPR / Cas12a fluorescence detection system uses TwistDx TwistAmp TM Basic Kit RPA enzyme and matching reagents, DEPC treated water are purchased from Shengong Biotechnology Co., Ltd. (Shanghai), and Cas12a is purchased from NEB Company in the United States.

[0060] 3. The detection method comprises the following steps (1) Extracting the genomic DNA of the sample to be tested; (2) Using the DNA template of step (1) and the RPA primer in Example 1 to perform isothermal amplification reaction to obtain RPA amplification product; Isothermal amplification reaction system and conditions: add RPA buffer (Rehydration buffer) 29.5 μL, DEPC-H2O or ddH2O 11.2 μL, 10 μmol / L Pi-RPA-F primer 2.4 μL, 10 μmol / L Pi-RPA-R primer 2.4 μL, 2 μL DNA, 280 mmol / L magnesium acetate 2.5 μL in the tube containing RPA enzyme lyophilized powder (TwistDx TwistAmp TM Basic Kit), mix well, and then place the reaction tube at 35 ℃ and heat for 15 min.

[0061] (3) Using the above Pi-crRNA guide sequence to prepare Cas12a / crRNA complex, and then adding ssDNA reporter molecule and RPA amplification product of step (2) to perform cleavage reaction in CRISPR / Cas12a system to obtain cleavage product; The RPA-CRISPR / Cas12a amplification system is as follows: DEPC-H2O 14.8 μL, NEBuffer (10x) 2 μL, 5 μmol / L Cas12a enzyme protein 0.2 μL, 5 μmol / L FQ-DNA 0.8 μL, 0.01 mmol / L Pi-crRNA 0.2 μL, RPA amplification product in Example 1 2 μL, and after uniform mixing, the reaction tube is placed at 37 ℃, and heated for 15-30 min. Then color development is observed under blue light (wavelength 470 nm).

[0062] (4) The lysis product of step (3) is subjected to color development under blue light (wavelength 470 nm) or naked eye observation. If the lysis product has no fluorescence brightness under blue light irradiation or no brightness under naked eye observation, it indicates that the sample to be tested is not / does not contain Brassica vegetable black stem rot fungus. If the lysis product has fluorescence brightness under blue light irradiation or brightness under naked eye observation, it indicates that the sample to be tested is / is contains Brassica vegetable black stem rot fungus. The detection results are shown in Figure 2 The PCR tubes from left to right represent the detection results of the strains with serial numbers 1-13 in Table 1. As can be seen from the figure, the amplification products of Brassica vegetable black stem rot fungi with serial numbers 7, 8 and 9 show obvious fluorescence signals.

[0063] Example 3 Optimization of RPA-CRISPR / Cas12a fluorescence detection system and sensitivity detection The RPA-CRISPR / Cas12a fluorescence detection system established in Example 2 is further optimized to make the whole reaction more rapid and efficient, and to reduce the cost.

[0064] 1. Optimization of the concentration of fluorescent reporter molecule FQ-DNA The concentration of fluorescent reporter molecule FQ-DNA is optimized. The reporter molecule concentration is set to 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L and 800 nmol / L, and the other conditions remain unchanged. The sterile water is used as a negative control. The RPA-CRISPR / Cas12a fluorescence detection system established in Example 2 is used to determine the concentration of fluorescent reporter molecule FQ-DNA.

[0065] The detection results of different concentrations of fluorescent reporter molecules are shown in Figure 3 The PCR tubes from left to right represent the reporter molecule concentrations of 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L and 800 nmol / L.

[0066] The results show that when the final concentration of the CRISPR / Cas12a fluorescent reporter molecule reaches 200 nmol / L, a clear fluorescence signal can be observed, and when the final concentration of the reporter molecule is greater than 200 nmol / L, the fluorescence gradually weakens. Therefore, the final concentration of 200 nmol / L of the CRISPR / Cas12a fluorescent reporter molecule is the optimal choice.

[0067] 2. Optimization of isothermal amplification reaction time Using the RPA-CRISPR / Cas12a fluorescent detection system established in Example 2, the RPA reaction time was unchanged, and was 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, and the other conditions were unchanged. Sterile water was used as a negative control.

[0068] 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.

[0069] The results show that the longer the reaction time, the stronger the fluorescence intensity. When the reaction time reaches 10 min, fluorescence can be detected. With the increase of CRISPR reaction time, the fluorescence intensity increases. When the reaction time is 20 min, the fluorescence intensity is significantly increased. Therefore, 20 min can be selected as the CRISPR / Cas12a fluorescent detection reaction time.

[0070] 3. Sensitivity experiment Using the RPA-CRISPR / Cas12a fluorescent detection system established in Example 2, the concentration of the fluorescent reporter molecule FQ-DNA and the isothermal amplification reaction time were adjusted to the results of 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. Each concentration gradient was set with 3 repeats.

[0071] The sensitivity detection results are shown in Figure 5 The PCR tubes from left to right represent DNA concentrations of 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, and 0.01 pg, respectively.

[0072] The results show that when the concentration of the genomic DNA of the cruciferous vegetable black stem rot fungus is greater than or equal to 10 pg, a fluorescence signal can be produced. This indicates that the sensitivity of the CRISPR / Cas12a fluorescent detection method is 10 pg.

[0073] In summary, the total reaction time is within 35 min (RPA reaction for 15 min, cleavage for 20 min), and the sensitivity of RPA-CRISPR / Cas12a fluorescence detection of Alternaria solani in cruciferous vegetables is 10 pg.

[0074] Example 4 Establishment of RPA-CRISPR / Cas12a lateral flow test strip detection system 1. In the 357 bp fragment amplified by RPA in Example 1, the crRNA guide sequence Pi-crRNA was designed by combining the CRISPR / Cas12a recognition site PAM, and the sequence was 5'-UAAUUUCUACUCUUGUAGAUGGGGAUCCAAUUGGUGGGCAAG-3'.

[0075] The nucleotide sequence of the ssDNA reporter molecule is LF-DNA: 5'-(6-FAM) TTTTTTTTTT (Biotin)-3'.

[0076] The above sequence is synthesized by Beijing Tianyihuiyuan Biotechnology Co., Ltd.

[0077] 2. The RPA-CRISPR / Cas12a lateral flow test strip detection system uses TwistAmp TM Basic Kit RPA enzyme and matching reagent from Shengong Biotechnology Co., Ltd. (Shanghai), Cas12a enzyme protein from NEB M0653T EnGen Lba Cas12a (Cpf1) of the United States.

[0078] 3. The detection method comprises the following steps: (1) Extracting the genomic DNA of the sample to be tested; (2) Using the DNA template of step (1) and the RPA primer in Example 1 to perform isothermal amplification reaction to obtain RPA amplification product; Isothermal amplification reaction system and conditions: add RPA buffer (Rehydration buffer) 29.5 μL, DEPC-H2O or ddH2O 11.2 μL, 10 μmol / L Pi-RPA-F primer 2.4 μL, 10 μmol / L Pi-RPA-R primer 2.4 μL, 2 μL DNA, 280 mmol / L magnesium acetate 2.5 μL in the tube containing RPA enzyme lyophilized powder (TwistAmp TM Basic Kit), mix well, and then place the reaction tube at 35 ℃ for heating reaction for 15 min.

[0079] (3) Using the above-mentioned Pi-crRNA guide sequence to prepare a Cas12a / crRNA complex, then adding an ssDNA reporter molecule and the RPA amplification product of step (2), and performing a cleavage reaction in a CRISPR / Cas12a system to obtain a cleavage product; Isothermal amplification reaction system and conditions: DEPC-H2O or ddH2O 11.6 μL, NEBuffer (10x) 2 μL, 5 μmol / L Cas12a enzyme protein 0.2 μL, 5 μmol / L LF-DNA 4 μL, 0.01 mmol / L Pi-crRNA 0.2 μL, 2 μL RPA amplification product obtained in step (2), mix uniformly, then place the reaction tube at 37 ℃, heat reaction for 15-30 min. After the reaction is completed, add 80 μL DEPC-H2O and mix uniformly.

[0080] (4) The cleavage product obtained in step (3) is subjected to colorimetric detection using a lateral flow test strip. If a red band appears on the test strip of the sample to be tested or red bands appear at the positions of the test strip of the sample to be tested and the negative sample control line, it indicates that the sample to be tested is / contains Brassica vegetable black stem rot fungus. If no red band appears on the test strip and a red band appears on the control line, it indicates that the sample to be tested is not / does not contain Brassica vegetable black stem rot fungus.

[0081] The detection results are shown in Figure 6 , and the test strips from left to right represent the detection results of the strains with serial numbers 1-13 in Table 1. As can be seen from the figure, the amplification products of Brassica vegetable black stem rot fungi with serial numbers 7, 8 and 9 show detection bands.

[0082] Example 5 Optimization of RPA-CRISPR / Cas12a lateral flow test strip detection system and sensitivity detection The RPA-CRISPR / Cas12a lateral flow test strip detection system established in Example 4 is further optimized to make the entire reaction more rapid and efficient, while reducing costs.

[0083] 1. Optimization of lateral flow test strip reporter molecule LF-DNA concentration The concentration of the lateral flow test strip reporter molecule LF-DNA is optimized, and the reporter molecule concentration is set to 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, 800 nmol / L, and other conditions remain unchanged. The sterile water is used as a negative control. The RPA-CRISPR / Cas12a lateral flow test strip detection system established in Example 4 is used to determine the concentration of the test strip reporter molecule LF-DNA.

[0084] The detection results of test strip reporter molecules with different concentrations are as followsFigure 7 The test strips from left to right represent the reporter molecule concentrations of 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, 800 nmol / L, respectively.

[0085] The results show that when the reporter molecule concentration is 50 nmol / L in the 100 μL test strip detection system, the test band (T line) can detect the weak color reaction, and when the reporter molecule concentration is 50-200 nmol / L, the color reaction gradually becomes obvious, but when the reporter molecule concentration is greater than 200 nmol / L, the color reaction becomes weak. Therefore, the final concentration of the CRISPR-Cas12a test strip reporter molecule can be 100 nmol / L.

[0086] 2. Isothermal amplification reaction time optimization The RPA-CRISPR / Cas12a lateral flow test strip detection system established in Example 4 was used, the RPA reaction time was unchanged, and was 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.

[0087] The detection results of different reaction times are shown in Figure 8 The test strips from left to right represent the amplification reaction times of 5 min, 10 min, 15 min, 20 min, and 30 min, respectively.

[0088] The results show that when the reaction time reaches 20 min, the product can be detected obviously. Therefore, 20 min can be selected as the CRISPR / Cas12a test strip detection reaction time.

[0089] 3. Sensitivity experiment 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 to the results of Example 5 optimization. The template DNA concentration of the target species was diluted by 10 times, and 6 concentrations were set, including 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, and 0.01 pg. Each concentration gradient was set with 3 repeats.

[0090] The sensitivity detection results are shown in Figure 9 The test strips from left to right represent the 6 DNA concentrations of 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, and 0.01 pg, respectively.

[0091] The results show that the weak detection signal can be generated when the genomic DNA concentration of the Alternaria brassicae is 10 pg, and the detection signal is enhanced with the increase of the concentration.

[0092] In summary, the total reaction time is within 35 min (RPA reaction for 15 min, cleavage for 20 min), and the detection sensitivity of the lateral flow test strip for the Alternaria brassicae is 10 pg.

[0093] 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, such as the preparation of RPA reaction and CRISPR / Cas12a reaction reagents into freeze-dried powder, which is obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present application are within the scope of the present application.

Claims

1. A crRNA for detecting Plasmodiophora brassicae Plenodomus lingam characterized in that, The guide sequence of the crRNA is Pi-crRNA: 5'-UAAUUUCUACUCUUGUAGAUGGGGAUCCAAUUGGUGGGCAAG-3'.

2. A cruciferous vegetable Phytophthora blight detection reaction system characterized by, The RPA amplification reaction system and the Cas12a detection reaction system; the Cas12a detection reaction system comprises the crRNA of claim 1.

3. The detection reaction system according to claim 2, wherein The RPA amplification reaction system comprises an RPA primer pair with sequences shown in SEQ ID NO: 1-2.

4. The kit for visualizing detection of Xanthomonas campestris pv. campestris based on RPA-CRISPR / Cas12a system, characterized in that, The RPA amplification reaction system comprises an RPA primer pair with sequences shown in SEQ ID NO: 1-2.

5. The kit of claim 4, wherein The kit further comprises RPA enzyme, RPA buffer, magnesium acetate, NEBuffer, Cas12a enzyme protein, ssDNA reporter molecule; The ssDNA reporter molecule is FQ-DNA or LF-DNA; The reporter molecule FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3', based on fluorescence detection method; The reporter molecule LF-DNA; 5'-(6-FAM) TTTTTTTTTT (Biotin)-3', based on lateral flow test strip detection method.

6. The kit of claim 4 or 5 for detecting Plenodomus lingam.

7. A method for visual detection of Altemaria brassicicola based on RPA-CRISPR / Cas12a system, characterized in that, The kit comprises the following steps: S1. Extracting the genomic DNA of the sample to be tested; S2. Using the RPA primer pair shown in SEQ ID NO: 1-2 to perform RPA isothermal amplification reaction with the total DNA of step S1 as the template, to obtain RPA amplification products; S3. Using the RPA amplification products of step S2 as the template, adding the Cas12a detection reaction system to perform CRISPR reaction; The Cas12a detection reaction system comprises the crRNA of claim 1, Cas12a enzyme protein and ssDNA reporter molecule; The ssDNA reporter molecule is FQ-DNA or LF-DNA; The reporter molecule FQ-DNA: 5'-(6-FAM) TTATT (BHQ1)-3', based on fluorescence detection method; The 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 step S3 with the reporter molecule FQ-DNA is subjected to color development under blue light with a wavelength of 470 nm or naked eye observation, if the reaction product has no fluorescence brightness or naked eye observation brightness under blue light irradiation, it indicates that the sample to be tested does not contain Plenodomus lingam, if the reaction product has fluorescence brightness or naked eye observation brightness under blue light irradiation, it indicates that the sample to be tested contains Plenodomus lingam; Visual lateral flow test strip detection reaction: the reaction product obtained by adding the reporter molecule LF-DNA in step S3 is detected by using a lateral flow test strip for color development. If a red band appears on the test strip of the sample to be tested or red bands appear at the positions of the test strip of the sample to be tested and the negative sample control line, it indicates that the sample to be tested contains Brassica vegetable black leg fungus. If no red band appears on the test strip and a red band appears on the control line, it indicates that the sample to be tested does not contain Brassica vegetable black leg fungus.

8. The method of claim 7, wherein, 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; The amplification reaction conditions are: 35℃, 15-30 min.

9. The method of claim 7, wherein, 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; The CRISPR reaction conditions are: 37℃, 15-30 min.

10. The method of claim 7, wherein, In the CRISPR reaction system used in step S3, the concentrations of crRNA, Cas12a enzyme protein and ssDNA reporter molecule LF-DNA are 100-300 nmon / L, 50-200 nmon / L and 50-800 nmon / L, respectively; The CRISPR reaction conditions are: 37℃, 15-30 min.

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