Method and kit for visual detection of entomophthora sylvatica

Abstract

The application provides a method and a kit for visual detection of soil-dwelling Cystothrix. The kit comprises specific RPA primer pairs and crRNA for detecting soil-dwelling Cystothrix. The soil-dwelling Cystothrix is detected by double specificity of RPA amplification and crRNA recognition, so that the plant or sample infected with soil-dwelling Cystothrix can be found as early as possible for quarantine treatment. The application has the characteristics of convenient operation, less time consumption, high sensitivity (the DNA detection limit can reach 0.01 pg) and strong specificity, and the whole process is carried out in an environment of 37-39 DEG C, which can effectively break away from the dependence on large instruments. The visual detection kit provided by the application can quickly and accurately screen the samples containing soil-dwelling Cystothrix at quarantine ports, prevent the spread of the pathogenic bacteria with the import and export and transportation of seeds and other propagation materials, and has important significance for the detection, prevention and control of plant pathogenic soil-dwelling Cystothrix and the promotion of the healthy development of agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a method and kit for visually detecting soil-dwelling thorny spores. Background Technology

[0002] Soil-dwelling Echinococcus Setophoma terrestris (HN Hansen) Gruyter (also known as: pink root rot fungus of onions, Latin synonym) Pyrenochaeta terrestris , Phoma terrestris *Allium chinense* is an important plant pathogenic fungus that can infect a wide variety of plants, including but not limited to onions, garlic, and leeks; Cucurbitaceae (cucurbits, squash); Fabaceae (peas, alfalfa, cowpeas); Poaceae (wheat, oats, rice, corn); and Solanaceae (tomatoes), seriously threatening crop production. This pathogen is mainly distributed in Senegal, South Africa, Canada, the United States, Mexico, Argentina, Brazil, Venezuela, Japan, Vietnam, India, the Netherlands, and Australia, with only localized reports in China.

[0003] Currently, with increasingly close trade relations between countries and a growing circulation of various agricultural products and commodities, the possibility of this pathogen entering and establishing itself in my country is high. For example, onions are one of the preferred vegetables for seafarers and are also an important plant product for quarantine of inbound and outbound ships. The *Echinochloa crus-galli* carried by onions poses a risk of cross-border transmission via sea transport. Early warning, rapid detection, risk analysis, and scientific control of invasive pests are the core elements of preventing the spread of alien species, with pest identification being the primary step. Effective identification of *Echinochloa crus-galli* plays a crucial role in controlling its damage and promptly blocking its spread. Therefore, establishing specific, sensitive, rapid, and effective molecular detection methods for the pathogen *Echinochloa crus-galli* is of great significance for the rapid customs clearance of imported plants and plant products and for early field disease diagnosis.

[0004] In the past, a fluorescence quantitative PCR detection method has been established in China to detect soil-dwelling Echinochloa crus-galli. However, this method has high requirements for the instruments and the technical skills of the testing personnel. At the same time, the experimental equipment is expensive and it is difficult to achieve on-site testing. Summary of the Invention

[0005] The purpose of this invention is to provide a method and kit for visually detecting soil-dwelling Echinochloa crus-galli.

[0006] To achieve the objective of this invention, in a first aspect, this invention provides a crRNA for detecting soil-dwelling Echinochloa crus-galli, wherein the guide sequence of the crRNA is St-crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCGAUCGUAGCCCGUUGUACUGG-3' (SEQ ID NO:3).

[0007] In a second aspect, the present invention provides a detection reaction system for soil-dwelling spiny spores, including an RPA amplification reaction system and a Cas12a detection reaction system; the Cas12a detection reaction system includes crRNA as shown in SEQ ID NO:3.

[0008] Furthermore, the RPA amplification reaction system includes RPA primer pairs with sequences as shown in SEQ ID NO:1-2.

[0009] Thirdly, the present invention provides a kit for visual detection of soil-dwelling Echinochloa crus-galli based on RPA and Cas12a, comprising crRNA as shown in SEQ ID NO:3 and RPA primer pairs as shown in SEQ ID NO:1-2.

[0010] Furthermore, the kit also includes the Cas12a enzyme protein and an ssDNA reporter molecule.

[0011] The ssDNA is either FQ-DNA or LF-DNA;

[0012] FQ-DNA: 5'-FAM-TTATT-BHQ1-3' (fluorescence detection method);

[0013] LF-DNA: 5'-FAM-TTTTTTTTTT-Biotin-3' (lateral flow test strip method).

[0014] Fourthly, the present invention provides the application of the kit in the detection of soil-dwelling Echinochloa crus-galli.

[0015] Fifthly, the present invention provides a method for visually detecting soil-dwelling thorny spores, comprising the following steps:

[0016] S1. Extract genomic DNA from the sample to be tested;

[0017] 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;

[0018] 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;

[0019] The Cas12a detection reaction system includes the crRNA, Cas12a enzyme protein, and ssDNA reporter molecule.

[0020] The ssDNA is either FQ-DNA or LF-DNA;

[0021] FQ-DNA: 5'-FAM-TTATT-BHQ1-3' (fluorescence detection method);

[0022] LF-DNA: 5'-FAM-TTTTTTTTTT-Biotin-3' (lateral flow test strip method);

[0023] S4. Visualization of Fluorescence Reaction: The reaction product from step S3 is developed under blue light (wavelength 470 nm) or observed with the naked eye. If the reaction product shows no fluorescence under blue light or no brightness when observed with the naked eye, it indicates that the sample does not contain *Echinochloa crus-galli*. If the reaction product shows fluorescence under blue light or brightness when observed with the naked eye, it indicates that the sample contains *Echinochloa crus-galli*. Alternatively...

[0024] Visualized lateral flow test strip detection reaction: The reaction product obtained in step S3 is 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 if 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 contains Echinococcus solaniae. 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 does not contain Echinococcus solaniae.

[0025] Furthermore, in the RPA isothermal amplification reaction system used in step S2, the concentrations of both the forward and reverse primers are 480 nmol / L;

[0026] The amplification reaction conditions were: 39℃, 20 min.

[0027] Furthermore, in the CRISPR reaction system used in step S3, the concentrations of crRNA, Cas12a enzyme protein, and ssDNA reporter molecule FQ-DNA are 200 nmol / L, 100 nmol / L, and 200 nmol / L, respectively.

[0028] The CRISPR reaction conditions are: 37℃, 5-10 min.

[0029] Furthermore, in the CRISPR reaction system used in step S3, the concentrations of crRNA, Cas12a enzyme protein, and ssDNA reporter molecule LF-DNA are 200 nmol / L, 100 nmol / L, and 100 nmol / L, respectively.

[0030] The CRISPR reaction conditions are: 37℃, 5-10 min.

[0031] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0032] (I) The visual detection kit for *Echinochloa crus-galli* provided by this invention involves first performing RPA amplification on the sample, then guiding the CRISPR-Cas12a system under the guidance of a crRNA-guided sequence to recognize and bind to the RPA amplification product and cleave the target double-stranded DNA to activate non-specific nuclease function. Subsequently, the ssDNA reporter molecule in the system is cleaved to obtain lysate products, and finally, the lysate products are detected by colorimetric analysis. This invention's RPA-CRISPR / Cas12a technology for detecting *Echinochloa crus-galli* specifically detects the smear through both RPA amplification and crRNA recognition, exhibiting high specificity and sensitivity (DNA detection limit as low as 0.01 pg). This allows for early detection of infected plants or samples for quarantine treatment. This invention is characterized by convenient operation, short processing time, high sensitivity, and high specificity. The entire process is conducted at 37-39℃, effectively eliminating reliance on large laboratory instruments.

[0033] (II) The visualization detection kit provided by this invention can quickly and accurately screen samples containing soil-dwelling spiny spores at quarantine ports, preventing the spread of pathogens through the import, export, and transportation of seeds and other propagation materials. It is of great significance for the detection and control of plant pathogenic soil-dwelling spiny spores and for promoting the healthy development of agricultural production, and has great application and promotion value. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the results of RPA primer specificity in a preferred embodiment of the present invention;

[0035] Figure 2 This is a schematic diagram of the primer and reporter molecule specificity results of the RPA combined with CRISPR / Cas12a fluorescence detection method in a preferred embodiment of the present invention;

[0036] Figure 3 This is a schematic diagram illustrating the optimized reporter molecule concentration of the RPA combined with the CRISPR / Cas12a fluorescence detection method in a preferred embodiment of the present invention.

[0037] Figure 4 This is a schematic diagram illustrating the time optimization visualization results of the RPA combined with CRISPR / Cas12a fluorescence detection method in a preferred embodiment of the present invention.

[0038] Figure 5 This is a schematic diagram of the sensitivity detection results of the RPA combined with CRISPR / Cas12a fluorescence detection method in a preferred embodiment of the present invention.

[0039] Figure 6This describes the 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 (T: test band; C: control line).

[0040] 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 in the figure (T: test band; C: control line).

[0041] Figure 8 Different reaction times (T: test strip; C: control line) are shown in the RPA-CRISPR / Cas12a lateral flow test strip detection system in a preferred embodiment of the present invention.

[0042] 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

[0043] This invention provides primers, probes, detection methods, and kits for the visual detection of soil-dwelling Echinochloa crus-galli based on RPA amplification combined with the CRISPR / Cas12a system. These methods are easy to operate, rapid, sensitive, and provide accurate identification results.

[0044] The present invention adopts the following technical solution:

[0045] This invention utilizes species of the genus *Echinochloa* ( Setophoma The ITS sequences of *S. spp.* 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.

[0046] A kit for detecting soil-dwelling Echinochloa crus-galli includes RPA enzyme lyophilized powder, RPA primers, crRNA guide sequence, Cas12a enzyme, and ssDNA reporter molecule;

[0047] The nucleotide sequences of the RPA primers are: St-RPA-F: 5'-GGGCGTCTTGTCGTATTACGACTCGCCTTAAATT-3' (SEQ ID NO:1), St-RPA-R: 5'-CCCTACCTGATCCGAGGTCAAACGTGGTAAATGT-3' (SEQ ID NO:2), and the crRNA guide sequence is St-crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCGAUCGUAGCCCGUUGUACUGG-3' (SEQ ID NO:3).

[0048] 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 soil-dwelling Echinochloa crus-galli in the target system by 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.

[0049] The nucleotide sequences of the ssDNA reporter molecules are FQ-DNA: 5'-(FAM)TTATT(BHQ1)-3', LF-DNA: 5'-(FAM)TTTTTTTTTT(Biotin)-3'.

[0050] The application of the specific primer pair or the primer-probe combination includes any of the following:

[0051] (1) Identify whether the sample to be tested is a soil-dwelling spiny spore;

[0052] (2) Preparation of a kit for identifying soil-dwelling thorny spores;

[0053] (3) Detect whether the sample to be tested contains soil-dwelling spiny spores;

[0054] (4) Prepare a kit for detecting whether the sample to be tested contains soil-dwelling spiny spores.

[0055] The present invention provides a kit for identifying soil-dwelling thorny spores, comprising the specific primer pair or the primer-probe combination.

[0056] The kit also includes one or more of the following: Rehydration buffer, DEPC water, magnesium acetate, NE buffer, lateral flow test strips, and positive template.

[0057] A method for detecting soil-dwelling Echinochloa crus-galli using visual fluorescence detection or visual lateral flow test strips includes the following steps:

[0058] S1. Extract genomic DNA from the sample to be tested;

[0059] S2. RPA amplification: Using the DNA from step S1 as a template, an isothermal amplification reaction was performed using the RPA primers described above to obtain the RPA amplification product;

[0060] S3. CRISPR / Cas system reaction detection: Cas12a / crRNA complex was prepared using the above crRNA guide sequence, and then ssDNA reporter molecule and RPA amplification product from step S2 were added. The lysis reaction was carried out in the CRISPR / Cas12a system to obtain lysis products.

[0061] S4. Visualization of fluorescence reaction: The lysis products from step S3 are developed under blue light (wavelength 470 nm) or observed with the naked eye. If the lysis products show no fluorescence under blue light or no brightness when observed with the naked eye, it indicates that the sample to be tested is not / does not contain Echinochloa crus-galli. If the lysis products show fluorescence under blue light or brightness when observed with the naked eye, it indicates that the sample to be tested is / contains Echinochloa crus-galli.

[0062] Visualized lateral flow test strip detection reaction: 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 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 is / contains *Echinochloa crus-galli*. 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 is not / does not contain *Echinochloa crus-galli*.

[0063] The system and conditions for the isothermal amplification reaction described in step S2 are as follows: 50 μL system, i.e., in a tube containing RPA enzyme lyophilized powder (TwistDx TwistAmp). TM Add 29.5 μL of rehydration buffer, 11.2 μL of DEPC-H2O, 2.4 μL of 10 μmol / L St-RPA-F primer, 2.4 μL of 10 μmol / L St-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 39 °C for 20 min.

[0064] The lysis reaction system and conditions for the fluorescence detection method described in step S3 are as follows: a 20 μL system, namely, 14.4 μL DEPC-H2O, 2 μL NEBuffer (10×), 0.4 μL 5 μmol / L Cas12a, 0.8 μL 5 μmol / L FQ-DNA, 0.4 μL 0.01 mmol / L St-crRNA, and 2 μL of the RPA amplification product obtained in step S2. After mixing thoroughly, the reaction tube is placed at 37°C and heated for 30 min. Then, colorimetric observation is performed under blue light (wavelength 470 nm) (see step S4).

[0065] The lysis reaction system and conditions for the lateral flow test strip detection method described in step S3 are as follows: 100 μL system, i.e., 13.2 μL DEPC-H2O, 2 μL NEBuffer (10×), 0.4 μL 5 μmol / L Cas12a, 2 μL 5 μmol / L LF-DNA, 0.4 μL 0.01 mmol / L St-crRNA, and 2 μL RPA amplification product obtained in step S2. After mixing thoroughly, the reaction tube is placed at 37 ℃ and heated for 20 min. After the reaction is complete, 80 μL DEPC-H2O is added and mixed thoroughly (see step S4).

[0066] In the isothermal amplification reaction described in step S3, the minimum detection sensitivity of the DNA is 0.01 pg.

[0067] In the isothermal amplification reaction system of the fluorescence detection method described in step S3, the final concentration of the CRISPR-Cas12a reporter molecule FQ-DNA is 200 nmol / L, which is the optimal choice. The nucleotide sequence of the ssDNA reporter molecule is FQ-DNA: 5'-(FAM)TTATT(BHQ1)-3'.

[0068] In the isothermal amplification reaction system of the lateral flow test strip detection method described in step S3, the final concentration of the CRISPR-Cas12a reporter molecule LF-DNA is 100 nmol / L, which is the optimal choice. The nucleotide sequence of the ssDNA reporter molecule is LF-DNA: 5'-(FAM)TTTTTTTTTT(Biotin)-3'.

[0069] In the isothermal amplification reaction described in step S3, the concentrations of the Cas12a protein and crRNA are 50 nM and 100 nM, or 100 nM and 200 nM, respectively.

[0070] In this invention, the sample to be tested can be a plant sample, a pure culture of fungi, a soil sample, etc., and the plant sample can specifically be a leaf, stem, root, fruit, etc.

[0071] The principle of the above detection method is that CRISPR-Cas12a recognizes a specific target in the RPA amplification product under the guidance of crRNA. CRISPR-Cas12a, crRNA and target sequence molecules combine to form a complex. The complex cuts the ssDNA reporter molecule in the detection system. After the probe molecule is cut, a large amount of fluorescence is generated and it can be detected.

[0072] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0073] Example 1: RPA primer design and amplification system determination

[0074] ITS sequences of *Echinochloa crus-galli* and its closely related fungi (Table 1) were obtained from the NCBI database. Sequence alignment and analysis were performed. Based on the specific target sequence of *Echinochloa crus-galli*, a series of primer pairs that meet the RPA primer design principles were designed. After a series of preliminary tests and screenings, the following primers (SEQ ID NO: 1-2) were determined:

[0075] Forward primer St-RPA-F sequence: 5'- GGGCGTCTTGTCGTATTACGACTCGCCTTAAATT-3';

[0076] Reverse primer St-RPA-R sequence: 5'-CCCTACCTGATCCGAGGTCAAACGTGGTAAATGT-3'

[0077] The amplified fragment size is 145 bp;

[0078] After determining the optimal primer pairs, an RPA amplification system was established, and the amplification temperature, amplification time, and other conditions were optimized.

[0079] 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 St-RPA-F, 2.4 μL of 10 μmol / L St-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 39 °C for 20 min.

[0080] Gel electrophoresis results showed that the primer pair St-RPA-F / St-RPA-R had good specificity for *Echinochloa crus-galli*. Figure 1 ). Figure 1 Samples numbered 1-9 correspond to strains numbered 1-9 in Table 1, with only *Echinochloa crus-galli* showing amplified bands.

[0081] Of the nine 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 of the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences.

[0082] Table 1. Soil-dwelling Echinococcus and its closely related fungi used for specific detection ( Setophoma = S. )

[0083] ;

[0084] Example 2: Establishment of RPA-CRISPR / Cas12a fluorescence detection system

[0085] 1. In the 145bp fragment amplified by RPA in Example 1, the CRISPR-Cas12a recognition site PAM was combined to design the crRNA guide sequence St-crRNA, whose sequence is 5'-UAAUUUCUACUAAGUGUAGAUCGAUCGUAGCCCGUUGUACUGG-3' (SEQ ID NO:3).

[0086] The nucleotide sequence of the ssDNA reporter molecule is FQ-DNA: 5'-(FAM)TTATT(BHQ1)-3'.

[0087] The above sequence was synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd.

[0088] 2. The RPA-CRISPR / Cas12a fluorescence detection system was developed using TwistDx's TwistAmp. TM The RPA enzyme and matching reagents in the Basic Kit, the DEPC-treated water were purchased from Sangon Biotech (Shanghai) Co., Ltd., and Cas12a was purchased from NEB Corporation, USA.

[0089] 3. The detection method includes the following steps:

[0090] (1) Extract genomic DNA from the sample to be tested;

[0091] (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;

[0092] 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 St-RPA-F primer, 2.4 μL of 10 μmol / L St-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 39 °C for 20 min.

[0093] (3) Prepare a Cas12a / crRNA complex using the above St-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;

[0094] The RPA-CRISPR / cas12a amplification system consisted of: 14.4 μL DEPC-H2O, 2 μL NEBuffer (10×), 0.4 μL 5 μmol / L Cas12a, 0.8 μL FQ-DNA, 0.4 μL 0.01 mmol / L St-crRNA, and 2 μL of the RPA amplification product from Example 1. After thorough mixing, the reaction tube was placed at 37 °C and heated for 30 min. The reaction was then observed under blue light (wavelength 470 nm).

[0095] (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 soil-dwelling echinoderms. 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 soil-dwelling echinoderms.

[0096] 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 1-9 in Table 1. It can be seen from the figure that only the amplification products of Echinochloa crus-galli, numbered 8 and 9, show obvious fluorescent signals.

[0097] Example 3: Optimization and Sensitivity Detection of the RPA-CRISPR / cas12a Fluorescence Detection System

[0098] 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.

[0099] 1. Optimization of the concentration of fluorescent reporter molecule FQ-DNA

[0100] The concentration of the fluorescent reporter molecule FQ-DNA was optimized by setting the reporter molecule concentrations to 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, and 800 nmol / L, while keeping other conditions unchanged. Sterile water was used as a negative control. The concentration of the fluorescent reporter molecule FQ-DNA was determined using the RPA-CRISPR / cas12a fluorescence detection system established in Example 2.

[0101] Detection results of fluorescent reporter molecules at different concentrations, as follows 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.

[0102] The results showed that the higher the concentration of the CRISPR-Cas12a fluorescent reporter molecule, the stronger the fluorescence intensity. A significant fluorescence signal was observed when the final concentration of the reporter molecule reached above 100 nmol / L, and the fluorescence intensity was extremely pronounced at a final concentration of 800 nmol / L. To balance cost and fluorescence intensity, a final concentration of 200 nmol / L for the CRISPR-Cas12a fluorescent reporter molecule was chosen as the optimal choice.

[0103] 2. Optimization of isothermal amplification reaction time

[0104] The RPA-CRISPR / cas12a fluorescence detection system established in Example 2 was used. The RPA reaction time remained unchanged at 20 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. Other conditions remained unchanged. Sterile water was used as a negative control.

[0105] Detection results at different reaction times, such as 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.

[0106] The results showed that the longer the reaction time, the stronger the fluorescence intensity. Fluorescence could be detected when the reaction time reached 5 min; the fluorescence intensity increased with increasing CRISPR reaction time. A significant fluorescence intensity was observed at a reaction time of 10 min. Therefore, 5-10 min can be selected as the CRISPR-Cas12a fluorescence detection reaction time.

[0107] 3. Sensitivity Experiment

[0108] The RPA-CRISPR / cas12a fluorescence detection system established in Example 2 was used, in which the concentration of the fluorescent reporter molecule FQ-DNA and the isothermal amplification reaction time were adjusted to the results optimized in Example 3. The template DNA concentration of the target species was serially diluted 10-fold, with a total of 6 concentrations: 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, and 0.01 pg. Three replicates were set for each concentration gradient.

[0109] Sensitivity test results are as follows Figure 5 The PCR tubes, from left to right, represent the concentrations of fluorescent reporter molecules: 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, and 0.01 pg, respectively.

[0110] The results showed that a concentration of 1 pg of genomic DNA from *Echinochloa crus-galli* produced a significant fluorescent signal, while a concentration of 0.01 pg produced a detectable weak fluorescent signal. This indicates that the sensitivity of the CRISPR-Cas12a fluorescence detection method is 0.01 pg.

[0111] In summary, with a total reaction time of less than 30 min (RPA reaction 20 min, cutting 10 min), the fluorescence detection sensitivity of *Echinochloa crus-galli* RPA-CRISPR / cas12a was 0.01 pg.

[0112] Example 4: Establishment of the RPA-CRISPR / Cas12a lateral flow test strip detection system

[0113] 1. In the 145bp fragment amplified by RPA in Example 1, the CRISPR-Cas12a recognition site PAM was combined to design the crRNA guide sequence St-crRNA, whose sequence is 5'-UAAUUUCUACUAAGUGUAGAUCGAUCGUAGCCCGUUGUACUGG-3'.

[0114] The nucleotide sequence of the ssDNA reporter molecule is LF-DNA: 5'-(FAM)TTTTTTTTTT(Biotin)-3'.

[0115] The above sequence was synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd.

[0116] 2. The RPA-CRISPR / Cas12a lateral flow test strip detection system uses TwistDx's TwistAmp... TM The RPA enzyme and matching reagents in the Basic Kit, the DEPC-treated water were purchased from Sangon Biotech (Shanghai) Co., Ltd., and Cas12a was purchased from NEB Corporation, USA.

[0117] 3. The detection method includes the following steps:

[0118] (1) Extract genomic DNA from the sample to be tested;

[0119] (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;

[0120] 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 St-RPA-F primer, 2.4 μL of 10 μmol / L St-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 39 °C for 20 min.

[0121] (3) Prepare a Cas12a / crRNA complex using the above St-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;

[0122] Isothermal amplification reaction system and conditions: DEPC-H2O 13.2 μL, NEBuffer (10×) 2 μL, 5 μmol / L Cas12a 0.4 μL, 5 μmol / L LF-DNA 2 μL, 0.01 mmol / L St-crRNA 0.4 μL, and 2 μL of the RPA amplification product obtained in step S2. After mixing thoroughly, the reaction tube was placed at 37 ℃ and heated for 20 min. After the reaction was complete, 80 μL of DEPC-H2O was added and mixed thoroughly.

[0123] (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 Echinochloa crus-galli. 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 Echinochloa crus-galli.

[0124] Test results as follows Figure 6 As shown in the figure, the test strips represent the detection results of bacterial species numbered 1-9 in Table 1 from left to right. It can be seen from the figure that only the amplification products of Echinochloa crus-galli, numbered 8 and 9, show the detection band.

[0125] Example 5: Optimization and Sensitivity Detection of the RPA-CRISPR / cas12a Lateral Flow Test Strip System

[0126] The RPA-CRISPR / cas12a lateral flow test strip detection system established in Example 4 was further optimized to make the entire reaction faster and more efficient, while reducing costs.

[0127] 1. Optimization of LF-DNA concentration in lateral flow test strips

[0128] The concentration of the reporter molecule LF-DNA on the lateral flow test strip was optimized by setting the reporter molecule concentrations to 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, and 800 nmol / L, while keeping other conditions unchanged. Sterile water was used as a negative control. The concentration of the reporter molecule LF-DNA on the test strip was determined using the RPA-CRISPR / cas12a lateral flow test strip detection system established in Example 4.

[0129] Test strips of different concentrations report molecular detection results, such as Figure 7 The test strips, from left to right, represent the reported molecule concentrations of 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, and 800 nmol / L, respectively.

[0130] The results showed that the test bands (T lines) were all distinct when the reporter molecule concentration in the 100 μL test strip detection system was 50-800 nmol / L.

[0131] 2. Optimization of isothermal amplification reaction time

[0132] The RPA-CRISPR / cas12a lateral flow test strip detection system established in Example 4 was used. The RPA reaction time remained unchanged at 20 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. Other conditions remained unchanged. Sterile water was used as a negative control.

[0133] Detection results at different reaction times, such as Figure 8 The test strips, from left to right, represent amplification reaction times of 5 min, 10 min, 15 min, 20 min, and 30 min, respectively.

[0134] The results showed that a detectable product was produced after 5 minutes of reaction, exhibiting a weak test band (T line). Therefore, 5-10 minutes can be selected as the reaction time for CRISPR / Cas12a.

[0135] 3. Sensitivity Experiment

[0136] The RPA-CRISPR / cas12a lateral flow test strip detection system established in Example 4 was used, in which the concentration of the reporter molecule LF-DNA and the isothermal amplification reaction time were adjusted to the results optimized in Example 5. The template DNA concentration of the target species was serially diluted 10-fold, with a total of 6 concentrations: 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.

[0137] Sensitivity test results are as follows Figure 9 The test strips, from left to right, represent six concentrations: 1 ng, 0.1 ng, 10 pg, 1 pg, 0.1 pg, and 0.01 pg.

[0138] The results showed that a genomic DNA concentration of 0.01 pg of *Echinochloa crus-galli* produced a weak detection signal, which increased with increasing concentration.

[0139] In summary, with a total reaction time of less than 30 min (20 min for RPA reaction and 10 min for cutting), the detection sensitivity of the soil-dwelling Echinochloa crus-galli RPA-CRISPR / cas12a lateral flow test strip is 0.01 pg.

[0140] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A reaction system for detecting terrestrial spiny spores, 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 Solanum sphaerocephala; the sequence of the crRNA is: 5'-UAAUUUCUACUAAGUGUAGAUCGAUCGUAGCCCGUUGUACUGG-3'; The RPA amplification reaction system includes RPA primer pairs with sequences as shown in SEQ ID NO:1-2.

2. A kit for visually detecting soil-dwelling thorny spores, 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 is either FQ-DNA or LF-DNA; FQ-DNA: 5'-FAM-TTATT-BHQ1-3', used for fluorescence detection; LF-DNA: 5'-FAM-TTTTTTTTTT-Biotin-3', used for lateral flow test strip detection.

4. The application of the kit described in claim 2 or 3 in the detection of soil-dwelling Echinochloa crus-galli.

5. A method for visually detecting soil-dwelling thorny spores, 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 comprises the crRNA, Cas12a enzyme protein, and ssDNA reporter molecule as described in claim 3; The ssDNA is either FQ-DNA or LF-DNA; FQ-DNA: 5'-FAM-TTATT-BHQ1-3', used for fluorescence detection; LF-DNA: 5'-FAM-TTTTTTTTTT-Biotin-3', used for lateral flow test strip detection method; S4. Visualization of Fluorescence Reaction: The reaction product from step S3 is developed under blue light at a wavelength of 470 nm or observed with the naked eye. If the reaction product shows no fluorescence under blue light or no brightness when observed with the naked eye, it indicates that the sample does not contain *Echinochloa crus-galli*. If the reaction product shows fluorescence under blue light or brightness when observed with the naked eye, it indicates that the sample contains *Echinochloa crus-galli*. Alternatively... Visualized lateral flow test strip detection reaction: The reaction product obtained in step S3 is detected by color development using a lateral flow test strip. If red bands appear at 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 Echinococcus solaniae. If no red bands appear at the test bands but red bands appear at the control line, it indicates that the sample to be tested does not contain Echinococcus solaniaeae.

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 nmol / L. The amplification reaction conditions were: 39℃, 20 min.

7. 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 FQ-DNA were 200 nmol / L, 100 nmol / L, and 200 nmol / L, respectively. The CRISPR reaction conditions are: 37℃, 5-10 min.

8. 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 200 nmol / L, 100 nmol / L, and 100 nmol / L, respectively. The CRISPR reaction conditions are: 37℃, 5-10 min.

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